Yersinia pestis antigens, vaccine compositions, and related methods

ABSTRACT

The present invention provides antigens and vaccines useful in prevention of infection by  Yersinia pestis . The present invention provides pharmaceutical compositions of such antigens and/or vaccines. The present invention provides methods for the production of  Y. pestis  protein antigens in plants, as well as methods for their use in the treatment and/or prevention of  Y. pestis  infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/668,258, filed Oct. 22, 2010, which is a National Stage applicationunder 35 U.S.C. §371 of International Application No. PCT/US2008/069860having an International Filing Date of Jul. 11, 2008, which claims thebenefit of priority of U.S. Provisional Patent Application Ser. No.60/949,115, filed Jul. 11, 2007, each of which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Historically plague has been a major infectious disease afflicting humanpopulations, leading to millions of deaths. The etiologic agent ofplague is Yersinia pestis and infection with this pathogen can developinto a highly contagious pneumonic disease with almost 100% lethality.Continued outbreaks of plague, along with the suitability of Y. pestisfor weaponization has heightened interest in developing a vaccine.Currently, there is no safe and effective vaccine against Y. pestis.

Thus, there is a need to provide sources of vaccines and antigens forproduction of vaccines. Improved vaccine design and development, as wellas methods of making and using such compositions of matter are neededwhich provide inexpensive and highly accessible sources of suchtherapeutic and/or prophylactic compositions.

SUMMARY OF THE INVENTION

The present invention provides Yersinia pestis antigens and vaccinecomponents produced in plants. The present invention provides one ormore Y. pestis antigens generated as a fusion with a thermostableprotein (e.g. lichenase). The invention provides vaccine compositionscontaining Y. pestis antigens. Furthermore, the invention provides Y.pestis vaccines comprising at least two different Y. pestis antigens, insome embodiments, compositions in accordance with the invention includeone or more plant components. Still further provided are methods forproduction and use of antigen and vaccine compositions in accordancewith the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a-1 x. Alignment of amino acid sequences of LcrV protein frommultiple Y. pestis strains. CLUSTAL W multiple sequence alignments ofLcrV amino acid sequences from 64 different Y. pestis strains (GenBankaccession numbers NP_(—)863514.1; NP_(—)783665.1; NP_(—)052392.1;AAK69213.1; AAN37531.1; AAD16815.1; YP_(—)068466.1; CAF25400.1;P0C556.1; NP_(—)995380.1; AAS58571.1; ZP_(—)02318603.1;ZP_(—)02314654.1; ZP_(—)02314147.1; ZP_(—)02314145.1; ZP_(—)02307430.1;EDR63976.1; EDR60080.1; EDR55652.1; EDR55650.1; EDR55212.1;ZP_(—)02240571.1; EDR48750.1; EDR41684.1; ZP_(—)02232674.1;ZP_(—)02228629.1; ZP_(—)02223652.1; EDR37557.1; EDR30648.1;YP_(—)001604463.1; ABX88711.1; CAB54908.1; ABF48194.1; ABF48193.1;ABF48192.1; ABF48191.1; ABF48190.1; ABF48189.1; NP_(—)395165.1;YP_(—)001293940.1; NP_(—)857946.1; NP_(—)857751.1; ABR68791.1;ABR68790.1; ABR68789.1; ABR68788.1; ABR 14856.1; AAC69799.1; AAC62574.1;AAF64077.1; A4TSQ1.1; YP_(—)001004069.1; CAL10039.1; P23994.1;AAA27645.1; AAF64076.1; YP_(—)636823.1; ABG16274. I; ABP42325.1;YP_(—)001154615.1; ABB16313.1; YP_(—)001874676.1; ACC91219.1;ABI97154.1) aligned with the sequence of LcrV that was used in theproduction of antigen constructs in the Exemplification (“LcrV.pro”).

FIG. 2. In vitro characterization of plant-produced Y. pestis antigens.Plant-produced LicKM (Lane 1), LicKM-LcrV (Lane 2), and LicKM-F1 (Lane3) were analyzed by SDS-PAGE followed by Coomassie staining (A) andimmunoblotting using rabbit polyclonal anti-LicKM (B), mouse monoclonalanti-LcrV (C), and mouse monoclonal anti-F1 (D) antibodies.

FIG. 3. Antibody responses elicited by plant-produced plague vaccineantigens and their protective efficacy against Y. pestis challenge.Serum samples were tested by ELISA for the presence of LcrV− (A and C)and F1− (B and D) specific IgG (A and C) and IgA (B and D). Data arerepresented as average titer a: standard deviation. Animals werechallenged with 100×LD₅₀ aerosolized Y. pestis, and the percentsurvivors for each experimental group were graphed over time (E).

FIG. 4. Production of LcrV-F1-LicKM fusion protein. Lanes 1-4: CoomassieBrilliant Blue staining Lanes 5-8: western blot using α-lichenaseantibody. Lanes 1 and 5: molecular weight markers. Lane 2: bovine serumalbumin. Lanes 3 and 7: LcrV-LicKM fusion. Lanes 4 and 8: LcrV-F1-LicKMfusion protein, wherein LcrV is inserted into the loop region of LicKM,and F1 is fused to LicKM as a C-terminal fusion. Lane 6: LicKM-LFfusion.

FIG. 5. Antibody responses elicited by plant-produced plague vaccineantigens and their protective efficacy against Y. pestis challenge.Serum samples were tested by ELISA for the presence of LcrV− (A and C)and F1− (B and D) specific IgG. Data are represented as averagetiter±standard deviation. The graphs shown in (A) and (B) differ fromthose in (C) and (D) only in the scale of the Y-axis.

FIG. 6. Survival of groups of female cynomolgus monkeys vaccinated threetimes subcutaneously or subcutaneously as a priming vaccination followedby twice intranasal vaccinations. The two plant-produced antigens (i.e.,F1 and LcrV) were presented to monkeys as a mixture ofindependently-derived fusion products with LicKM or as a double fusionproduct (LicKM-F1-LcrV). Group 1 ( ••••••) a control group with LicKM,received 125 μg LicKM plus two adjuvants. Group 2 ( - - - ) monkeysreceived 250 μg LicKM-FI and LicKM-LcrV mixture plus two adjuvants. Bothgroups were vaccinated subcutaneously, thrice. Group 3 (

), a control group, received 125 μg LicKM plus two adjuvants as asubcutaneous priming dose followed by two intranasal doses withoutadjuvant at two week intervals. Group 4 ( - - - - ) received 250 μgLicKM-F1 and LicKM-LcrV mixture plus two adjuvants as a subcutaneouspriming dose followed by two intranasal doses without adjuvant at twoweek intervals. Group 5 (

) received 250 μg LicKM-F1-LcrV double fusion product plus two antigensthree times subcutaneously at two week intervals. On post-infection day0 (Study Day 40), all monkeys were exposed to multiple LD₅₀ inhalationdose of Y. pestis CO92. Monkeys were followed up to post-infection day14 (Study Day 54).

DEFINITIONS

Amino acid: As used herein, term “amino acid,” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally-occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a D-aminoacid; in some embodiments, an amino acid is an L-amino acid. “Standardamino acid” refers to any of the twenty standard L-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and/or substitutions. Amino acids, including carboxy-and/or amino-terminal amino acids in peptides, can be modified bymethylation, amidation, acetylation, and/or substitution with otherchemical groups that can change the peptide's circulating half-lifewithout adversely affecting their activity. Amino acids may participatein a disulfide bond. The term “amino acid” is used interchangeably with“amino acid residue,” and may refer to a free amino acid and/or to anamino acid residue of a peptide. It will be apparent from the context inwhich the term is used whether it refers to a free amino acid or aresidue of a peptide.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal,genetically-engineered animal, and/or a clone.

Antibody: As used herein, the term “antibody” refers to anyimmunoglobulin, whether natural or wholly or partially syntheticallyproduced. All derivatives thereof which maintain specific bindingability are also included in the term. The term also covers any proteinhaving a binding domain which is homologous or largely homologous to animmunoglobulin binding domain. Such proteins may be derived from naturalsources, or partly or wholly synthetically produced. An antibody may bemonoclonal or polyclonal. An antibody may be a member of anyimmunoglobulin class, including any of the human classes: IgG, IgM, IgA,IgD, and IgE. As used herein, the terms “antibody fragment” or“characteristic portion of an antibody” are used interchangeably andrefer to any derivative of an antibody which is less than full-length.In general, an antibody fragment retains at least a significant portionof the full-length antibody's specific binding ability. Examples ofantibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2,scFv, Fv, dsFv diabody, and Fd fragments. An antibody fragment may beproduced by any means. For example, an antibody fragment may beenzymatically or chemically produced by fragmentation of an intactantibody and/or it may be recombinantly produced from a gene encodingthe partial antibody sequence. Alternatively or additionally, anantibody fragment may be wholly or partially synthetically produced. Anantibody fragment may optionally comprise a single chain antibodyfragment. Alternatively or additionally, an antibody fragment maycomprise multiple chains which are linked together, for example, bydisulfide linkages. An antibody fragment may optionally comprise amultimolecular complex. A functional antibody fragment typicallycomprises at least about 50 amino acids and more typically comprises atleast about 200 amino acids.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, and/or 3′ end formation);(3) translation of an RNA into a polypeptide or protein; (4)posttranslational modification of a polypeptide or protein.

Gene: As used herein, the term “gene” has its meaning as understood inthe art. It will be appreciated by those of ordinary skill in the artthat the term “gene” may include gene regulatory sequences (e.g.,promoters, enhancers, etc.) and/or intron sequences. It will further beappreciated that definitions of gene include references to nucleic acidsthat do not encode proteins but rather encode functional RNA moleculessuch as tRNAs. For the purpose of clarity we note that, as used in thepresent application, the term “gene” generally refers to a portion of anucleic acid that encodes a protein; the term may optionally encompassregulatory sequences, as will be clear from context to those of ordinaryskill in the art. This definition is not intended to exclude applicationof the term “gene” to non-protein-coding expression units but rather toclarify that, in most cases, the term as used in this document refers toa protein-coding nucleic acid.

Gene product: As used herein, the term “gene product” or “expressionproduct” generally refers to an RNA transcribed from the gene (pre-and/or post-processing) or a polypeptide (pre- and/or post-modification)encoded by an RNA transcribed from the gene.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 95%, or about 99% identical. In some embodiments,polymeric molecules are considered to be “homologous” to one another iftheir sequences are at least about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, or about 99% similar.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twonucleic acid sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced ha one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, or about 100%of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using the algorithm of Meyers and Miller(CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGNprogram (version 2.0) using a PAM 120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. The percent identity between twonucleotide sequences can, alternatively, be determined using the GAPprogram in the GCG software package using an NWSgapdna.CMP matrix.

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of theother components with which they were initially associated. In someembodiments, isolated agents are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, substantially 100%, or 100% pure. Asused herein, a substance is “pure” if it is substantially free of othercomponents. As used herein, the term “isolated cell” refers to a cellnot contained in a multi-cellular organism.

Nucleic acid: As used herein, the term “nucleic acid,” in its broadestsense, refers to any compound and/or substance that is or can beincorporated into an oligonucleotide chain. In some embodiments, anucleic acid is a compound and/or substance that is or can beincorporated into an oligonucleotide chain via a phosphodiester linkage.In some embodiments, “nucleic acid” refers to individual nucleic acidresidues (e.g. nucleotides and/or nucleosides). In some embodiments,“nucleic acid” refers to an oligonucleotide chain comprising individualnucleic acid residues. As used herein, the terms “oligonucleotide” and“polynucleotide” can be used interchangeably. In some embodiments,“nucleic acid” encompasses RNA as well as single mid/or double-strandedDNA and/or cDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,”and/or similar terms include nucleic acid analogs, i.e. analogs havingother than a phosphodiester backbone. For example, the so called“peptide nucleic acids,” which are known in the art and have peptidebonds instead of phosphodiester bonds in the backbone, are consideredwithin the scope of the present invention. The term “nucleotide sequenceencoding an amino acid sequence” includes all nucleotide sequences thatare degenerate versions of each other and/or encode the same amino acidsequence. Nucleotide sequences that encode proteins and/or RNA mayinclude introns. Nucleic acids can be purified from natural sources,produced using recombinant expression systems and optionally purified,chemically synthesized, etc. Where appropriate, e.g., in the case ofchemically synthesized molecules, nucleic acids can comprise nucleosideanalogs such as analogs having chemically modified bases or sugars,backbone modifications, etc. A nucleic acid sequence is presented in the5′ to 3′ direction unless otherwise indicated. The term “nucleic acidsegment” is used herein to refer to a nucleic acid sequence that is aportion of a longer nucleic acid sequence. In many embodiments, anucleic acid segment comprises at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, or moreresidues. In some embodiments, a nucleic acid is or comprises naturalnucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxyeytidine);nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine,C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine,C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, and 2-tltiocytidine); chemically modified bases;biologically modified bases (e.g., methylated bases); intercalatedbases; modified sugars (e.g., 2′-fluorofibose, ribose, 2′-deoxyribose,arabinose, and hexose); and/or modified phosphate groups (e.g.,phosphorothioates and 5′-N-phosphoramidite linkages). In someembodiments, the present invention may be specifically directed to“unmodified nucleic acids,” meaning nucleic acids (e.g. polynucleotidesand residues, including nucleotides and/or nucleosides) that have notbeen chemically modified in order to facilitate or achieve delivery.

Operably linked: As used herein, the term “operably linked” refers to arelationship between two nucleic acid sequences wherein the expressionof one of the nucleic acid sequences is controlled by, regulated by,modulated by, etc., the other nucleic acid sequence. For example, thetranscription of a nucleic acid sequence is directed by an operablylinked promoter sequence; post-transcriptional processing of a nucleicacid is directed by an operably linked processing sequence; thetranslation of a nucleic acid sequence is directed by an operably linkedtranslational regulatory sequence; the transport or localization of anucleic acid or polypeptide is directed by an operably linked transportor localization sequence; and the post-translational processing of apolypeptide is directed by an operably linked processing sequence. Anucleic acid sequence that is operably linked to a second nucleic acidsequence may be covalently linked, either directly or indirectly, tosuch a sequence, although any effective three-dimensional association isacceptable.

Portion: As used herein, the phrase a “portion” or “fragment” of asubstance, in the broadest sense, is one that shares some degree ofsequence and/or structural identity and/or at least one functionalcharacteristic with the relevant intact substance. For example, a“portion” of a protein or polypeptide is one that contains a continuousstretch of amino acids, or a collection of continuous stretches of aminoacids, that together are characteristic of a protein or polypeptide. Insome embodiments, each such continuous stretch generally will contain atleast about 2, about 5, about 10, about 15, about 20 or more aminoacids. In general, a portion is one that, in addition to the sequenceidentity specified above, shares at least one functional characteristicwith the relevant intact protein. In some embodiments, the portion maybe biologically active.

Protein: As used herein, the term “protein” refers to a polypeptide(i.e., a string of at least two amino acids linked to one another bypeptide bonds). Proteins may include moieties other than amino acids(e.g., may be glycoproteins, proteoglycans, etc.) and/or may beotherwise processed or modified. Those of ordinary skill in the art willappreciate that a “protein” can be a complete polypeptide chain asproduced by a cell (with or without a signal sequence), or can be acharacteristic portion thereof. Those of ordinary skill will appreciatethat a protein can sometimes include more than one polypeptide chain,for example linked by one or more disulfide bonds or associated by othermeans. Polypeptides may contain L-amino acids, D-amino acids, or bothand may contain any of a variety of amino acid modifications or analogsknown in the art. Useful modifications include, e.g., terminalacetylation, amidation, etc. in some embodiments, proteins may comprisenatural amino acids, non-natural amino acids, synthetic amino acids, andcombinations thereof. The term “peptide” is generally used to refer to apolypeptide having a length of less than about 100 amino acids.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which compositions in accordance with the invention may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans; insects;worms; etc.).

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of the disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with tile disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition may not exhibitsymptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition will develop the disease, disorder, and/or condition.In some embodiments, an individual who is susceptible to a disease,disorder, and/or condition will not develop the disease, disorder,and/or condition.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” of a therapeutic agent means anamount that is sufficient, when administered to a subject suffering fromor susceptible to a disease, disorder, and/or condition, to treat,diagnose, prevent, and/or delay the onset of the symptom(s) of thedisease, disorder, and/or condition.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that, when administered to a subject, has a therapeuticeffect and/or elicits a desired biological and/or pharmacologicaleffect.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a biologically active agentthat partially or completely alleviates, ameliorates, relives, inhibits,delays onset of, prevents, reduces severity of and/or reduces incidenceof one or more symptoms or features of a particular disease, disorder,and/or condition. Such treatment may be of a subject who does notexhibit signs of the relevant disease, disorder and/or condition and/orof a subject who exhibits only early signs of the disease, disorder,and/or condition. Alternatively or additionally, such treatment may beof a subject who exhibits one or more established signs of the relevantdisease, disorder and/or condition.

Vector: As used herein, “vector” refers to a nucleic acid molecule whichcan transport another nucleic acid to which it has been linked. In someembodiment, vectors can achieve extra-chromosomal replication and/orexpression of nucleic acids to which they are linked in a host cell suchas a eukaryotic and/or prokaryotic cell. Vectors capable of directingthe expression of operatively linked genes are referred to herein as“expression vectors.”

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to Yersinia pestis antigens useful in thepreparation of vaccines against Y. pestis infection, and fusion proteinscomprising such Y. pestis antigens operably linked a thermostableprotein (e.g. lichenase). The invention relates to methods of productionof provided antigens, including but not limited to, production in plantsystems. Further, the invention relates to vectors, fusion proteins,plant cells, plants and vaccine compositions comprising antigens andfusion proteins in accordance with the invention. Still further providedare methods of inducing immune response against Y. pestis infection in asubject comprising administering vaccine compositions in accordance withthe invention to a subject.

Yersinia pestis Antigens

Yersinia pestis (also known as Pasteurella pestis) is a Gram-negativerod-shaped bacterium belonging to the family Enterobacteriaceae. It is afacultative anaerobe with bipolar staining (giving it a safety pinappearance). Similar to other Yersinia members, it tests negative forurease, lactose fermentation, and indole. Y. pestis can infect humansand other animals. Human Y. pestis infection takes three main forms:pneumonic, septicemic, and bubonic. All three forms have beenresponsible for high mortality rates in epidemics throughout humanhistory, including the Black Death (a bubonic plague) that accounted forthe death of approximately one-third of the European population in 1347to 1353. During many of these epidemics, Y. pestis was transmitted byfleas infesting rats.

Three biovars of Y. pestis are known, each thought to correspond to oneof the historical pandemics of bubonic plague. Biovar Antigua is thoughtto correspond to the Plague of Justinian; it is not known whether thisbiovar also corresponds to earlier, smaller epidemics of bubonic plague,or whether these were even truly bubonic plague. Biovar Medievalis isthought to correspond to the Black Death. Biovar Orientalis is thoughtto correspond to the Third Pandemic and the majority of modem outbreaksof plague.

The complete genomic sequence is available for two of the threesub-species of Y. pestis: strain KIM (of biovar Medievalis) (Deng etal., 2002, J. Bacteriol., 184:4601-11; incorporated herein by reference)and strain CO92 (of biovar Orientalis, obtained from a clinical isolatein the United States) (Parkhill et al., 2001, Nature, 413:523-7;incorporated herein by reference). As of 2006, the genomic sequence of astrain of biovar Antigua has been recently completed (Chain et al.,2006, J. Bacteriol, 188:4453-63; incorporated herein by reference). Thechromosome of strain KIM is 4,600,755 base pairs (bp) long; thechromosome of strain CO92 is 4,653,728 bp long. Like its cousins Y.pseudotuberculosis and Y. enterocolitica, Y. pestis is host to theplasmid pCD1. In addition, it also hosts two other plasmids, pPCP1 andpMT1, which are not carried by the other Yersinia species. Together,these plasmids, and a pathogenicity island called HPI, encode severalproteins which are thought to cause pathogenesis. Among other things,these virulence factors are involved in bacterial adhesion and injectionof proteins into the host cell, invasion of bacteria into the host cell,and acquisition and binding of iron harvested from red blood cells.

Y. pestis is thought to be descendant from Y. pseudotuberculosis,differing only in the presence of specific virulence plasmids. Forexample, Y. pestis LcrV sequences are typically between about 90%-about100% identical (e.g., about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about100% identical) to LcrV sequences from Y. pseudotuberculosis. Y. pestisLcrV sequences are typically between about 90% about 100% identical(e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, or about 100% identical) toLcrV sequences from Y. enterocolitica. Thus, the present inventionencompasses the recognition that Y. pestis antigens may be useful forconferring protectivity and/or mounting an immune response againstmultiple Yersinia species, including Y. pestis, Y. pseudotuberculosis,and/or Y. enterocolitica.

The traditional first line treatment for Y. pestis has beenstreptomycin, chloramphenicol, tetracycline, and fluoroquinolones. Insome cases, doxycycline or gentamicin might be useful to treat Y. pestisinfection. Antibiotic treatment alone is insufficient for some patients,who may also require circulatory, ventilator, or renal support. Prior tothe present invention, no plant-produced Y. pestis vaccine has beenshown to be safe and effective in humans or non-human primates (Alvarezet al., 2006, Vaccine, 24:2477-90; and Santi et al., 2006, Proc. Natl.Acad. Sci., USA, 103:861-6; Williamson et al., Infect. Immun., 2005,73:3598-608; Anderson et al., 1996, Infect. Immun., 64:4580-5; Andrewset al., 1996, Infect. Immun., 64:2180-7; Williamson et al., 2000,Vaccine, 19:566-71; Williamson et al., 1995, FEMS Immunol. Med.Microbiol., 12:223-30; and Heath et al., 1998, Vaccine, 16:1131-7; allof which are incorporated herein by reference).

Y. pestis antigen proteins in accordance with the invention include anyimmunogenic protein or peptide capable of eliciting an immune responseagainst Y. pestis. Generally, immunogenic proteins of interest includeY. pestis antigens (e.g., Y. pestis proteins, fusion proteins, etc.),immunogenic portions thereof, or immunogenic variants thereof andcombinations of any of the foregoing.

Any Y. pestis protein can be produced and utilized as an antigen inaccordance with the present invention. Typically, Y. pestis proteins(i.e. full-length proteins, portions, fragments, and/or domains thereof,peptides, etc.) that are useful as antigens are not substantiallyidentical and/or homologous to proteins which are expressed by theanimal being vaccinated. In some embodiments, Y. pestis proteins areless than 90%, less than 80%, less than 70%, less than 60%, less than50%, less than 40%, less than 30%, less than 20%, or less than 10%identical and/or homologous to proteins which are expressed by theanimal being vaccinated. In some embodiments, a particular Y. pestisprotein may have portions and/or domains that are substantiallyidentical and/or homologous to proteins which are expressed by theanimal being vaccinated as well as portions and/or domains that are notsubstantially identical and/or homologous to proteins which areexpressed by the animal being vaccinated. In some embodiments, proteinsand/or peptides to be used in accordance with the present invention areprotein portions and/or domains that are not substantially identicaland/or homologous to proteins which are expressed by the animal beingvaccinated that have been separated and/or isolated from proteinportions and/or domains that are substantially identical and/orhomologous to proteins which are expressed by the animal beingvaccinated.

Y. pestis antigens for use in accordance with the present invention mayinclude full-length Y. pestis proteins or portions (i.e. fragments,domains, etc.) of Y. pestis proteins, and/or fusion proteins comprisingfull-length Y. pestis proteins or portions of Y. pestis proteins. Whereportions of Y. pestis proteins are utilized, whether alone or in fusionproteins, such portions retain immunological activity (e.g.,cross-reactivity with anti-Y, pestis antibodies). The present inventionrelates to two Y. pestis antigens that are of interest for developing avaccine against Y. pestis: the anti-phagocytic capsular envelopeglycoprotein (F1) and the low calcium-response V (LcrV) protein.Additional antigens (e.g., proteins, lipoproteins, glycoproteins,proteoglycans, and/or peptidoglycans associated with cell membranesand/or cell surfaces; surface antigens; periplasmic proteins; etc.) maybe useful in production of vaccines (e.g., combination vaccines) inorder to improve efficacy of immunoprotection.

Thus, the invention provides plant cells and/or plants expressing aheterologous protein, such as a Y. pestis antigen (e.g., Y. pestisprotein or a fragment thereof, a fusion protein comprising a Y. pestisprotein or portion thereof). A heterologous protein in accordance withthe invention can comprise any Y. pestis antigen of interest, including,but not limited to F1, LcrV, or fusion proteins, portions, orcombinations of F1, LcrV, a portion of F1, and/or a portion of LcrV. Insome embodiments, the invention provides plant cells and/or plantsexpressing a full-length heterologous protein. In some embodiments, theinvention provides plant cells and/or plants expressing a portion of aheterologous protein. In some embodiments, the invention provides plantcells and/or plants expressing multiple portions of a heterologousprotein. In some embodiments, such multiple portions are each producedfrom an individual vector. In some embodiments, such multiple proteinportions are tandemly expressed from the same vector (i.e. a“polytope”). In some embodiments, all of the multiple protein portionsof a polytope are identical to one another. In some embodiments, not allof the multiple protein portions are identical to one another.

Amino acid sequences of a variety of different Y. pestis proteins (e.g.,F1 and/or LcrV) are known in the art and are available in publicdatabases such as GenBank. In some embodiments, Y. pestis antigenscomprise F1 protein and/or a characteristic portion thereof. In someembodiments, F1 protein is variable at amino acid position 48. In someembodiments, the amino acid at position 48 is alanine 1n someembodiments, the amino acid at position 48 is serine. In someembodiments, multiple F1 protein variants differ only at position 48.

Exemplary full-length amino acid sequences for F1 protein which comprisean alanine at residue 48 include, but are not limited to, amino acidsequences as set forth in GenBank accession numbers NP_(—)395430;AAM94402.1; AAM94401.1; AAM94400.1; AAM94399.1; AAM94398.1; AAM94397.1;AAM94396.1; AAM94395.1; NP_(—)995523.1; AAS58714.1; CAA43966.1;NP_(—)857881.1; AAC82758.1; YP_(—)636639.1; and YP_(—)636755.1. In someembodiments, an F1 protein comprising an alanine at residue 48 may beobtained, isolated, purified, and/or derived from Y. pestis strainsCO92, Antigua, Microtus, Str.91001, KIM, Nepal 516, and/or FV1.

One exemplary full length protein sequence for F1 protein comprising analanine at position 48 is:

(SEQ ID NO: 1) 5′ MKKISVIAIALFGTIATANA ADLTASTTATATLVEPARITLTYKEG A PITIMDNGNIDTELLVGTLTLGGYKTGTTSTSVNFTDAAGDPMYLTFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDVVLATGSQDFFVRSIGSKGGKLAAGKYTDAVTVTVSNQ 3′.The bold, underlined sequence above (i.e., MKK . . . ANA) corresponds toa signal sequence. The bold, underlined alanine residue at position 48corresponds to a site of variability in the F1 protein.

In some embodiments, a Y. pestis antigen comprises an amino acidsequence which is about 60% identical, about 70% identical, about 80%identical, about 85% identical, about 90% identical, about 91%identical, about 92% identical, about 93% identical, about 94%identical, about 95% identical, about 96% identical, about 97%identical, about 98% identical, about 99% identical, or 100% identicalto SEQ ID NO: 1.

In some embodiments, a Y. pestis antigen comprises an amino acidsequence which comprises about 100 contiguous amino acids of SEQ IDNO: 1. In some embodiments, a Y. pestis antigen comprises an amino acidsequence which is about 60% identical, about 70% identical, about 80%identical, about 85% identical, about 90% identical, about 91%identical, about 92% identical, about 93% identical, about 94%identical, about 95% identical, about 96% identical, about 97%identical, about 98% identical, about 99% identical, or 100% identicalto a contiguous stretch of about 100 amino acids of SEQ ID NO: 1.

One exemplary full-length nucleotide sequence encoding F1 proteincomprising an alanine at position 48 corresponds to GenBank accessionnumber NC_(—)003134 (83368.85869):

(SEQ ID NO: 2) 5′ ATGAAAAAAATCAGTTCCGTTATCGCCATTGCATTATTTGGAACTATTGCAACTGCTAATGC G GCAGATTTAACTGCAAGCACCACTGCAACGGCAACTCTTGTTGAACCAGCCCGCATCACTCTTACATATAAGGAAGGC GCT CCAATTACAATTATGGACAATGGAAACATCGATACAGAATTACTTGTTGGTACGCTTACTCTTGGCGGCTATAAAACAGGAACCACTAGCACATCTGTTAACTTTACAGATGCCGCGGGTGATCCCATGTACTTAACATTTACTTCTCAGGATGGAAATAACCACCAATTCACTACAAAAGTGATTGGCAAGGATTCTAGAGATTTTGATATCTCTCCTAAGGTAAACGGTGAGAACCTTGTGGGGGATGACGTCGTCTTGGCTACGGGCAGCCAGGATTTCTTTGTTCGCTCAATTGGTTCCAAAGGCGGTAAACTTGCAGCAGGTAAATACACTGATGCTGTAACCGTAACC GTATCTAACCAATAA 3′.The bold, underlined sequence above (i.e., ATG . . . GCG) corresponds tothe nucleotide sequence encoding a signal sequence of F1 protein. Thebold, underlined codon (i.e., GCT) corresponds to the nucleotidesequence encoding the alanine residue at position 48.

In some embodiments, full length F1 protein does not comprise the signalsequence. One exemplary full length protein sequence for F1 proteincomprising an alanine at position 48 but not comprising a signalsequence is:

(SEQ ID NO: 3) 5′ADLTASTTATATLVEPARITLTYKEG A PITIMDNGNIDTELLVGTLTLGGYKTGTTSTSVNFTDAAGDPMYLTFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDVVLATGSQDFFVRSIGSKGGKLAAGKYTDAVTVTVSN Q 3′.The bold, underlined alanine residue at position 48 corresponds to asite of variability in the F1 protein.

In some embodiments, a Y. pestis antigen comprises an amino acidsequence which is about 60% identical, about 70% identical, about 80%identical, about 85% identical, about 90% identical, about 91%identical, about 92% identical, about 93% identical, about 94%identical, about 95% identical, about 96% identical, about 97%identical, about 98% identical, about 99% identical, or 100% identicalto SEQ ID NO: 3.

In some embodiments, a Y. pestis antigen comprises an amino acidsequence which comprises about 100 contiguous amino acids of SEQ ID NO:3. In some embodiments, a Y. pestis antigen comprises an amino acidsequence which is about 60% identical, about 70% identical, about 80%identical, about 85% identical, about 90% identical, about 91%identical, about 92% identical, about 93% identical, about 94%identical, about 95% identical, about 96% identical, about 97%identical, about 98% identical, about 99% identical, or 100% identicalto a contiguous stretch of about 100 amino acids of SEQ ID NO: 3.

One exemplary full-length nucleotide sequence encoding F1 proteincomprising an alanine at position 48 but not comprising a signalsequence is:

(SEQ ID NO: 4) 5′GCAGATTTAACTGCAAGCACCACTGCAACGGCAACTCTTGTTGAACCAGCCCGCATCACTCTTACATATAAGGAAGGC GCT CCAATTACAATTATGGACAATGGAAACATCGATACAGAATTACTTGTTGGTACGCTTACTCTTGGCGGCTATAAAACAGGAACCACTAGCACATCTGTTAACTTTACAGATGCCGCGGGTGATCCCATGTACTTAACATTTACTTCTCAGGATGGAAATAACCACCAATTCACTACAAAAGTGATTGGCAAGGATTCTAGAGATTTTGATATCTCTCCTAAGGTAAACGGTGAGAACCTTGTGGGGGATGACGTCGTCTTGGCTACGGGCAGCCAGGATTTCTTTGTI′CGCTCAATTGGTTCCAAAGGCGGTAAACTTGCAGCAGGTAAATACACTGATGCTGTAACCGTAACCGTATCTAACCAA TAA 3′.The bold, underlined codon (i.e., GCT) corresponds to the nucleotidesequence encoding the alanine residue at position 48.

Exemplary full-length amino acid sequences for F1 protein which comprisea serine at residue 48 include, but are not limited to, amino acidsequences as set forth in GenBank accession numbers YP_(—)093952,YP_(—)001154728.1, CAG27478.1, and/or ABP42491.1. In some embodiments,an F1 protein comprising a serine at residue 48 may be obtained,isolated, purified, and/or derived from Y. pestis strains Pestoides,Calif. 88-4125, and/or EV.

One exemplary full length protein sequence for F1 protein comprising aserine at position 48 is:

(SEQ ID NO: 5) 5′ MKKISSVIAIALFGTIATANA ADLTASTTATATLVEPARITLTYKEGSPITIMDNGNIDTELLVGTLTLGGYKTGTTSTSVNFTDAAGDPMYLTFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDVVLATGSQDFFVRSIGSKGGKLAAGKYTDAVTVTVSNQ 3′.The bold, underlined sequence above (i.e., MKK . . . ANA) corresponds toa signal sequence. The bold, underlined serine residue at position 48corresponds to a site of variability in the F1 protein.

In some embodiments, a Y. pestis antigen comprises an amino acidsequence which is about 60% identical, about 70% identical, about 80%identical, about 85% identical, about 90% identical, about 91%identical, about 92% identical, about 93% identical, about 94%identical, about 95% identical, about 96% identical, about 97%identical, about 98% identical, about 99% identical, or 100% identicalto SEQ ID NO: 5.

In some embodiments, a Y. pestis antigen comprises an amino acidsequence which comprises about 100 contiguous amino acids of SEQ ID NO:5. In some embodiments, a Y. pestis antigen comprises an amino acidsequence which is about 60% identical, about 70% identical, about 80%identical, about 85% identical, about 90% identical, about 91%identical, about 92% identical, about 93% identical, about 94%identical, about 95% identical, about 96% identical, about 97%identical, about 98% identical, about 99% identical, or 100% identicalto a contiguous stretch of about 100 amino acids of SEQ ID NO: 5.

One exemplary full-length nucleotide sequence encoding F1 proteincomprising a serine at position 48 corresponds to GenBank accessionnumber NC_(—)006323.1:

(SEQ ID NO: 6) 5′ ATGAAAAAAATCAGTTCCGTTATCGCCATTGCATTATTTGGAACTATTGCAACTGCTAATGCGG CAGATTTAACTGCAAGCACCACTGCAACGGCAACTCTTGTTGAACCAGCCCGCATCACTCTTACATATAAGGAAGGC TCT CCAATTACAATTATGGACAATGGAAACATCGATACAGAATTACTTGTTGGTACGCTTACTCTTGGCGGCTATAAAACAGGAACCACTAGCACATCTGTTAACTTTACAGATGCCGCGGGTGATCCCATGTACTTAACATTTACTTCTCAGGATGGAAATAACCACCAATTCACTACAAAAGTGATTGGCAAGGATTCTAGAGATTTTGATATCTCTCCTAAGGTAAACGGTGAGAACCTTGTGGGGGATGACGTCGTCTTGGCTACGGGCAGCCAGGATTTCTTTGTTCGCTCAATTGGTTCCAAAGGCGGTAAACTTGCAGCAGGTAAATACACTGATGCTGTAACCGTAACC GTATCTAACCAATAA 3′.The bold, underlined sequence above (i.e., ATG . . . GCG) corresponds tothe nucleotide sequence encoding a signal sequence of F1 protein. Thebold, underlined codon (i.e., TCT) corresponds to the nucleotidesequence encoding the serine residue at position 48.

In some embodiments, full length F1 protein does not comprise the signalsequence. One exemplary full length protein sequence for F1 proteincomprising an alanine at position 48 but not comprising a signalsequence is:

(SEQ ID NO: 7) 5′ADLTASTTATATLVEPARITLTYKEG S PITIMDNGNIDTELLVGTLTLGGYKTGTTSTSVNFTDAAGDPMYLTFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDWLATGSQDFFVRSIGSKGGKLAAGKYTDAVTVTVSNQ 3′.The bold, underlined serine residue at position 48 corresponds to a siteof variability in the F1 protein.

In some embodiments, a Y. pestis antigen comprises an amino acidsequence which is about 60% identical, about 70% identical, about 80%identical, about 85% identical, about 90% identical, about 91%identical, about 92% identical, about 93% identical, about 94%identical, about 95% identical, about 96% identical, about 97%identical, about 98% identical, about 99% identical, or 100% identicalto SEQ ID NO: 7.

In some embodiments, a Y. pestis antigen comprises an amino acidsequence which comprises about 100 contiguous amino acids of SEQ ID NO:7. In some embodiments, a Y. pestis antigen comprises an amino acidsequence which is about 60% identical, about 70% identical, about 80%identical, about 85% identical, about 90% identical, about 91%identical, about 92% identical, about 93% identical, about 94%identical, about 95% identical, about 96% identical, about 97%identical, about 98% identical, about 99% identical, or 100% identicalto a contiguous stretch of about 100 amino acids of SEQ ID NO: 7.

One exemplary full-length nucleotide sequence encoding F1 proteincomprising a serine at position 48 but not comprising a signal sequenceis:

(SEQ ID NO: 8) 5′CAGATTTAACTGCAAGCACCACTGCAACGGCAACTCTTGTTGAACCAGCCCGCATCACTCTTACATATAAGGAAGGC TCT CCAATTACAATTATGGACAATGGAAACATCGATACAGAATTACTTGTTGGTACGCTTACTCTTGGCGGCTATAAAACAGGAACCACTAGCACATCTGTTAACTTTACAGATGCCGCGGGTGATCCCATGTACTTAACATTTACTTCTCAGGATGGAAATAACCACCAATTCACTACAAAAGTGATTGGCAAGGATTCTAGAGATTTTGATATCTCTCCTAAGGTAAACGGTGAGAACCTTGTGGGGGATGACGTCGTCTTGGCTACGGGCAGCCAGGATTTCTFTGTTCGCTCAATTGGTTCCAAAGGCGGTAAACTTGCAGCAGGTAAATACACTGATGCTGTAACCGTAACCGTATCTAACCAATA A 3′.The bold, underlined codon (i.e., TCT) corresponds to the nucleotidesequence encoding the serine residue at position 48.

In certain embodiments, fun length F1 protein is utilized in vaccinecompositions in accordance with the invention. In some embodiments, oneor more portions and/or domains of F1 protein is used. In certainembodiments, two or three or more portions and/or domains are utilized,as one or more separate polypeptides or linked together in one or morefusion polypeptides.

In some embodiments, Y. pestis antigens comprise LcrV protein and/or acharacteristic portion thereof. Exemplary full-length amino acidsequences for LcrV protein include, but are not limited to, amino acidsequences as set forth in GenBank accession numbers NP_(—)863514.1;NP_(—)783665.1; NP_(—)052392.1; AAK69213.1; AAN37531.1; AAD16815.1;YP_(—)068466.1; CAF25400.1; P00556.1; NP_(—)995380.1; AAS58571.1;ZP_(—)02318603.1; ZP_(—)02314654.1; ZP_(—)02314147.1; ZP_(—)02314145.1;ZP_(—)02307430.1; EDR63976.1; EDR60080.1; EDR55652.1; EDR55650.1;EDR55212.1; ZP_(—)02240571.1; EDR48750.1; EDR41684.1; ZP_(—)02232674.1;ZP_(—)02228629.1; ZP_(—)02223652.1; EDR37557.1; EDR30648.1;YP_(—)001604463.1; ABX88711.1; CAB54908.1; ABF48194.1; ABF48193.1;ABF48192.1; ABF48191.1; ABF48190.1; ABF48189.1; NP_(—)395165.1;YP_(—)001293940.1; NP_(—)857946.1; NP_(—)857751.1; ABR68791.1;ABR68790.1; ABR68789.1; ABR68788.1; ABR14856.1; AAC69799.1; AAC62574.1;AAF64077.1; A4TSQ1.1; YP_(—)001004069.1; CAL10039.1; P23994.1;AAA27645.1; AAF64076.1; YP_(—)636823.1; ABG16274.1; ABP42325.1;YP_(—)001154615.1; ABB16313.1; YP_(—)001874676.1; ACC91219.1; and/orABI97154.1 (SEQ ID NOs: 38-102, respectively; see FIG. 1). In someembodiments, an LcrV protein may be obtained, isolated, purified, and/orderived from Y. pestis strains, for example, from Antigua strainE1979001, Antigua strain B42003004, Ulegeica, CA88-4125, KIM, Orientalisstrain MG05-1020, and/or Mediaevalis strain K1973002. FIG. 1 presentsmultiple LcrV protein variants from different Y. pestis strains alignedwith the sequence of LcrV that was used in the production of antigenconstructs in the Exemplification (“LcrV.pro,” SEQ ID NO: 103).

One exemplary full length protein sequence for LcrV protein is:

(SEQ ID NO: 9) 5′MIRAYEQNPQHFIEDLEKVRVEQLTGHGSSVLEELVQLVKDKNIDISIKYDPRKDSEVFANRVITDDIELLKKILAYFLPEDAILKGGHYDNQLQNGIKRVKEFLESSPNTQWELRAFMAVMHFSLTADRIDDDILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDKNLYGYTDEEIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNSYSYNKDNNELSHFATTCSDKSRPLNDLVSQKTTQLSDITSRFNSAIEALNRFIQKYDSVMQRLLDDTSGK 3′.

In some embodiments, a Y. pestis antigen comprises an amino acidsequence which is about 60% identical, about 70% identical, about 80%identical, about 85% identical, about 90% identical, about 91%identical, about 92% identical, about 93% identical, about 94%identical, about 95% identical, about 96% identical, about 97%identical, about 98% identical, about 99% identical, or 100% identicalto SEQ ID NO: 9.

In some embodiments, a Y. pestis antigen comprises an amino acidsequence which comprises about 100 contiguous amino acids of SEQ ID NO:9. In some embodiments, a Y. pestis antigen comprises an amino acidsequence which is about 60% identical, about 70% identical, about 80%identical, about 85% identical, about 90% identical, about 91%identical, about 92% identical, about 93% identical, about 94%identical, about 95% identical, about 96% identical, about 97%identical, about 98% identical, about 99% identical, or 100% identicalto a contiguous stretch of about 100 amino acids of SEQ ID NO: 9.

One exemplary full-length nucleotide sequence encoding LcrV protein is:

(SEQ ID NO: 10) 5′ATGATAAGGGCTTATGAACAAAATCCACAGCATTTTATTGAAGACCTAGAGAAAGTGCGAGTCGAACAGCTGACCGGCCATGGGTCGTCCGTTCTCGAAGAATTGGTGCAATTAGTTAAAGATAAAAACATCGATATTTCTATTAAGTACGACCCTAGGAAGGATTCTGAGGTATTTGCTAATAGAGTGATTACAGATGATATTGAATTACTAAAAAAGATATTGGCATACTTCCTTCCTGAGGATGCTATTCTTAAGGGTGGACACTATGACAATCAACTTCAAAACGGCATTAAGAGGGTTAAGGAGTTCCTCGAAAGCTCTCCAAATACTCAATGGGAGTTACGTGCTTTTATGGCTGTTATGCATTTTAGTCTGACAGCTGATCGAATTGATGATGATATTCTAAAGGTAATTGTAGATTCCATGAATCATCACGGTGACGCCAGGTCTAAGTTGCGTGAAGAGCTTGCTGAGTTGACTGCTGAACTGAAGATATATTCCGTGATACAGGCAGAAATTAACAAGCACTTATCATCTTCAGGAACTATTAATATTCACGATAAGTCTATTAATCTTATGGATAAAAACCTATACGGTTATACTGATGAGGAGATTTTCAAAGCTAGTGCGGAGTACAAAATATTAGAAAAGATGCCCCAAACTACTATACAGGTGGATGGGTCTGAAAAGAAGATTGTTTCTATCAAAGATTTCCTGGGTAGCGAAAACAAAAGAACGGGAGCACTTGGGAATCTCAAGAATTCTTATTCATATAACAAAGATAACAACGAGCTTTCACATTTCGCAACTACTTGTAGTGATAAGTCCAGACCACTCAACGATCTTGTATCACAAAAGACAACTCAATTGTCTGACATTACTTCTCGTTTCAACAGCGCTATTGAAGCACTTAATAGGTTCATTCAGAAGTACGATTCTGTGATGCAAAGATTGCTFGATGATACATCTGGAAAG 3′.

In certain embodiments, full length LcrV protein is utilized in vaccinecompositions in accordance with the invention. In some embodiments oneor more portions and/or domains of LcrV protein is used. In certainembodiments, two or three or more portions and/or domains are utilized,as one or more separate polypeptides or linked together in one or morefusion polypeptides.

In some embodiments, a Y. pestis antigen composition comprises a fusionprotein. In some embodiments, the fusion protein may contain two or moreidentical antigen proteins. In some embodiments, the fusion protein maycontain two or more distinct antigen proteins. In some embodiments, a Y.pestis antigen composition comprises a fusion of F1 and LcrV proteins.

In some embodiments, a fusion of F1 and LcrV proteins has an amino acidsequence that is identical to that set forth in SEQ ID NO: 27. In someembodiments, a Y. pestis antigen comprises an amino acid sequence whichis about 60% identical, about 70% identical, about 80% identical, about85% identical, about 90% identical, about 91% identical, about 92%identical, about 93% identical, about 94% identical, about 95%identical, about 96% identical, about 97% identical, about 98%identical, about 99% identical, or 100% identical to SEQ ID NO: 27.

In some embodiments, a Y. pestis antigen comprises an amino acidsequence which comprises about 100 contiguous amino acids of SEQ ID NO:27. In some embodiments, a Y. pestis antigen comprises an amino acidsequence which is about 60% identical, about 70% identical, about 80%identical, about 85% identical, about 90% identical, about 91%identical, about 92% identical, about 93% identical, about 94%identical, about 95% identical, about 96% identical, about 97%identical, about 98% identical, about 99% identical, or 100% identicalto a contiguous stretch of about 100 amino acids of SEQ ID NO: 27.

In some embodiments, a fusion of F1 and LcrV proteins comprises an aminoacid sequence that is identical to that set forth in SEQ ID NO: 33. Insome embodiments, a Y. pestis antigen comprises an amino acid sequencewhich is about 60% identical, about 70% identical, about 80% identical,about 85% identical, about 90% identical, about 91% identical, about 92%identical, about 93% identical, about 94% identical, about 95%identical, about 96% identical, about 97% identical, about 98%identical, about 99% identical, or 100% identical to SEQ ID NO: 33.

In some embodiments, a Y. pestis antigen comprises an amino acidsequence which comprises about 100 contiguous amino acids of SEQ ID NO:33. In some embodiments, a Y. pestis antigen comprises an amino acidsequence which is about 60% identical, about 70% identical, about 80%identical, about 85% identical, about 90% identical, about 91%identical, about 92% identical, about 93% identical, about 94%identical, about 95% identical, about 96% identical, about 97%identical, about 98% identical, about 99% identical, or 100% identicalto a contiguous stretch of about 100 amino acids of SEQ ID NO: 33.

In some embodiments, a fusion of F1 and LcrV proteins comprises an aminoacid sequence that is identical to that set forth in SEQ ID NO: 34. Insome embodiments, a Y. pestis antigen comprises an amino acid sequencewhich is about 60% identical, about 70%, identical, about 80% identical,about 85% identical, about 90% identical, about 91% identical, about 92%identical, about 93% identical, about 94% identical, about 95%identical, about 96% identical, about 97% identical, about 98%identical, about 99% identical, or 100% identical to SEQ ID NO: 34.

In some embodiments, a Y. pestis antigen comprises an amino acidsequence which comprises about 100 contiguous amino acids of SEQ ID NO:34. In some embodiments, a Y. pestis antigen comprises an amino acidsequence which is about 60% identical, about 70% identical, about 80%identical, about 85% identical, about 90% identical, about 91%identical, about 92% identical, about 93% identical, about 94%identical, about 95% identical, about 96% identical, about 97%identical, about 98% identical, about 99% identical, or 100% identicalto a contiguous stretch of about 100 amino acids of SEQ ID NO: 34.

In some embodiments, a fusion of F1 and LcrV proteins comprises aminoacid sequences that are identical to those set forth in SEQ ID NOs: 33and 34.

In some embodiments, a Y. pestis antigen comprises amino acid sequenceswhich are greater than 60% identical, greater than 70% identical,greater than 80% identical, greater than 85% identical, greater than 90%identical, greater than 91% identical, greater than 92% identical,greater than 93% identical, greater than 94% identical, greater than 95%identical, greater than 96% identical, greater than 97% identical,greater than 98% identical, greater than 99% identical, or 100%identical to SEQ ID NOs: 33 and 34.

In some embodiments, Y. pestis antigen comprises amino acid sequenceswhich comprise about 100 contiguous amino acids of each of SEQ ID NOs:33 and 34. In some embodiments, a Y. pestis antigen comprises amino acidsequences which are greater than 60% identical, greater than 70%identical, greater than 80% identical, greater than 85% identical,greater than 90% identical, greater than 91% identical, greater than 92%identical, greater than 93% identical, greater than 94% identical,greater than 95% identical, greater than 96% identical, greater than 97%identical, greater than 98% identical, greater than 99% identical, or100% identical to contiguous stretches of about 100 amino acids of eachof SEQ ID NOs: 33 and 34.

As exemplary antigens, we have utilized sequences from Y. pestis F1 andLcrV as described in detail herein. However, it will be understood byone skilled in the art that the methods and compositions provided hereinmay be adapted to utilize any Y. pestis sequences. It will also beunderstood by one skilled in the art that the methods and compositionsprovided herein may be adapted to utilize sequences of any Y. pestisspecies and/or subtype. Such variation is contemplated and encompassedwithin the methods and compositions provided herein.

Production of Yersinia pestis Antigens

In accordance with the present invention, Y. pestis antigens (includingY. pestis protein(s), portions, fragments, domains, variants, and/orfusions thereof) may be produced in any desirable system; production isnot limited to plant systems. Vector constructs and expression systemsare well known in the art and may be adapted to incorporate use of Y.pestis antigens provided herein. For example, Y. pestis antigens(including Y. pestis protein(s), portions, fragments, domains, variants,and/or fusions thereof) can be produced in known expression systems,including mammalian cell systems, transgenic animals, microbialexpression systems, insect cell systems, and plant systems, includingtransgenic and transient plant systems. Particularly where Y. pestisantigens are produced as fusion proteins, it may be desirable to producesuch fusion proteins in non-plant systems.

In some embodiments, Y. pestis antigens are desirably produced in plantsystems. Plants are relatively easy to manipulate genetically, and haveseveral advantages over alternative sources such as human fluids, animalcell lines, recombinant microorganisms and transgenic animals. Plantshave sophisticated post-translational modification machinery forproteins that is similar to that of mammals (although it should be notedthat there are some differences in glycosylation patterns between plantsand mammals). This enables production of bioactive reagents in planttissues. Also, plants can economically produce very large amounts ofbiomass without requiring sophisticated facilities. Thus, proteinproduction in plants typically requires a much lower capital investmentand cost-of-goods than protein production using other systems. Moreover,plants are not subject to contamination with animal pathogens. Likeliposomes and microcapsules, plant cells are expected to provideprotection for passage of antigen to the gastrointestinal tract. In manyinstances, production of proteins in plants leads to improved consumersafety.

Plants may be utilized for production of heterologous proteins via useof various production systems. One such system includes use oftransgenic/genetically-modified plants where a gene encoding targetproduct is permanently incorporated into the genome of the plant.Transgenic systems may generate crop production systems. A variety offoreign proteins, including many of mammalian origin and many vaccinecandidate antigens, have been expressed in transgenic plants and shownto have functional activity (Tacket et al., 2000, J. Infect. Dis.,182:302; and Thanavata et al., 2005, Proc. Natl. Acad. Sci., USA,102:3378; both of which are incorporated herein by reference).Additionally, administration of unprocessed transgenic plants expressinghepatitis B major surface antigen to non-immunized human volunteersresulted in production of immune response (Kapusta et al., 1999, FASEBJ., 13:1796; incorporated herein by reference).

One system for expressing polypeptides in plants utilizes plant viralvectors engineered to express foreign sequences (e.g., transientexpression). This approach allows for use of healthy non-transgenicplants as rapid production systems. Thus, genetically engineered plantsand plants infected with recombinant plant viruses can serve as “greenfactories” to rapidly generate and produce specific proteins ofinterest. Plant viruses have certain advantages that make themattractive as expression vectors for foreign protein production. Severalmembers of plant RNA viruses have been well characterized, andinfectious cDNA clones are available to facilitate genetic manipulation.Once infectious viral genetic material enters a susceptible host cell,it replicates to high levels and spreads rapidly throughout the entireplant. There are several approaches to producing target polypeptidesusing plant viral expression vectors, including incorporation of targetpolypeptides into viral genomes. One approach involves engineering coatproteins of viruses that infect bacteria, animals or plants to functionas carrier molecules for antigenic peptides. Such carrier proteins havethe potential to assemble and form recombinant virus-like particlesdisplaying desired antigenic epitopes on their surface. This approachallows for time-efficient production of vaccine candidates, since theparticulate nature of a vaccine candidate facilitates easy andcost-effective recovery from plant tissue. Additional advantages includeenhanced target-specific immunogenicity, the potential to incorporatemultiple vaccine determinants, and ease of formulation into vaccinesthat can be delivered nasally, orally or parenterally. As an example,spinach leaves containing recombinant plant viral particles carryingepitopes of virus fused to coat protein have generated immune responseupon administration (Modelska et al., 1998, Proc. Natl. Acad. Sci., USA,95:2481; and Yusibov et al., 2002, Vaccine, 19/20:3155; both of whichare incorporated herein by reference).

Plant Expression Systems

Any plant susceptible to incorporation and/or maintenance ofheterologous nucleic acid and capable of producing heterologous proteinmay be utilized in accordance with the present invention. In general, itwill often be desirable to utilize plants that are amenable to growthunder defined conditions, for example in a greenhouse and/or in aqueoussystems. It may be desirable to select plants that are not typicallyconsumed by human beings or domesticated animals and/or are nottypically part of the human food chain, so that they may be grownoutside without concern that expressed polynucleotide may be undesirablyingested. In some embodiments, however, it will be desirable to employedible plants. In particular embodiments, it will be desirable toutilize plants that accumulate expressed polypeptides in edible portionsof a plant.

Often, certain desirable plant characteristics will be determined by theparticular polynucleotide to be expressed. To give but a few examples,when a polynucleotide encodes a protein to be produced in high yield (aswill often be the case, for example, when antigen proteins are to beexpressed), it will often be desirable to select plants with relativelyhigh biomass (e.g., tobacco, which has additional advantages that it ishighly susceptible to viral infection, has a short growth period, and isnot in the human food chain). Where a polynucleotide encodes antigenprotein whose full activity requires (or is inhibited by) a particularpost-translational modification, the ability (or inability) of certainplant species to accomplish relevant modification (e.g., a particularglycosylation) may direct selection. For example, plants are capable ofaccomplishing certain post-translational modifications (e.g.,glycosylation), however, plants will not generate sialylation patternswhich are found in mammalian post-translational modification. Thus,plant production of antigen may result in production of a differententity than the identical protein sequence produced in alternativesystems.

In certain embodiments, crop plants, or crop-related plants areutilized. In certain specific embodiments, edible plants are utilized.

Plants for use in accordance with the present invention includeAngiosperms, Bryophytes (e.g., Hepaticae, Musci, etc.), Pteridophytes(e.g., ferns, horsetails, lycopods), Gymnosperms (e.g., conifers,cycase, Ginko, Gnetales), and Algae (e.g., Chlorophyceae, Phaeophyceae,Rbodophyceae, Myxophyceae, Xanthophyceae, and Euglenophyceae). Exemplaryplants are members of the family Leguminosae (Fabaceae; e.g., pea,alfalfa, soybean); Gramineae (Poaceae; e.g., corn, wheat, rice);Solanaceae, particularly of the genus Lycopersicon (e.g., tomato),Solanum (e.g., potato, eggplant), Capsium (e.g., pepper), or Nicotiana(e.g., tobacco); Umbelliferae, particularly of the genus Daucus (e.g.,carrot), Apium (e.g., celery), or Rutaceae (e.g., oranges); Compositae,particularly of the genus Lactuca (e.g., lettuce); Brassicaceae(Cruciferae), particularly of the genus Brassica or Sinapis. In certainaspects, plants in accordance with tile invention may be plants of theBrassica or Arabidopsis genus. Some exemplary Brassieaeeae familymembers include Brassica campestris, B. carinata, B. juncea, B. napus,B. nigra, B. oleraceae, B. tournifortii, Sinapis alba, and Raphanussativus. Some suitable plants that are amendable to transformation andare edible as sprouted seedlings include alfalfa, mung bean, radish,wheat, mustard, spinach, carrot, beet, onion, garlic, celery, rhubarb, aleafy plant such as cabbage or lettuce, watercress or cress, herbs suchas parsley, mint, or clovers, cauliflower, broccoli, soybean, lentils,edible flowers such as sunflower, peas, etc.

Introducing Vectors into Plants

In general, vectors may be delivered to plants according to knowntechniques. For example, vectors themselves may be directly applied toplants (e.g., via abrasive inoculations, mechanized spray inoculations,vacuum infiltration, particle bombardment, or electroporation).Alternatively or additionally, virions may be prepared (e.g., fromalready infected plants), and may be applied to other plants accordingto known techniques.

A wide variety of viruses are known that infect various plant species,and can be employed for polynucleotide expression according to thepresent invention (see, for example, in The Classification andNomenclature of Viruses, “Sixth Report of the International Committee onTaxonomy of Viruses” (Ed. Murphy et al.), Springer Verlag: New York,1995, the entire contents of which are incorporated herein by reference;Grierson et al., Plant Molecular Biology, Blackie, London, pp. 126-146,1984; Gluzman et al., Communications in Molecular Biology: ViralVectors, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp.172-189, 1988; and Mathew, Plant Viruses Online(http://image.fs.uidaho.edu/vide/). In certain embodiments, rather thandelivering a single viral vector to a plant cell, multiple differentvectors are delivered which, together, allow for replication (and,optionally cell-to-cell and/or long distance movement) of viralvector(s). Some or all proteins may be encoded by the genome oftransgenic plants. In certain aspects, described in further detailherein, these systems include one or more viral vector components.

Vector systems that include components of two heterologous plant virusesin order to achieve a system that readily infects a wide range of planttypes and yet poses little or no risk of infectious spread. An exemplarysystem has been described previously (see, e.g., PCT Publication WO00/25574 and U.S. Patent Publication 2005/0026291, both of which areincorporated herein by reference). As noted herein, in particularaspects of the invention, viral vectors are applied to plants (e.g.,plant, portion of plant, sprout, etc.), for example, throughinfiltration or mechanical inoculation, spray, etc. Where infection isto be accomplished by direct application of a viral genome to a plant,any available technique may be used to prepare the genome. For example,many viruses that are usefully employed in accordance with the presentinvention have ssRNA genomes, ssRNA may be prepared by transcription ofa DNA copy of the genome, or by replication of an RNA copy, either invivo or in vitro. Given the readily availability of easy-to-use in vitrotranscription systems (e.g., SP6, T7, reticulocyte lysate, etc.), andalso the convenience of maintaining a DNA copy of an RNA vector, it isexpected that ssRNA vectors will often be prepared by in vitrotranscription, particularly with T7 or SP6 polymerase.

In certain embodiments, rather than introducing a single viral vectortype into a plant, multiple different viral vectors are introduced. Suchvectors may, for example, trans-complement each other with respect tofunctions such as replication, cell-to-cell movement, and/or longdistance movement. Vectors may contain different polynucleotidesencoding Y. pestis antigen in accordance with the invention. Selectionfor plant(s) or portions thereof that express multiple polypeptidesencoding one or more Y. pestis antigen(s) may be performed as describedabove for single polynucleotides or polypeptides.

Plant Tissue Expression Systems

As discussed above, in accordance with the present invention, Y. pestisantigens may be produced in any desirable system. Vector constructs andexpression systems are well known in the art and may be adapted toincorporate use of Y. pestis antigens provided herein. For example,transgenic plant production is known and generation of constructs andplant production may be adapted according to known techniques in theart. In some embodiments, transient expression systems in plants aredesirable. Two of these systems include production of clonal roots andclonal plant systems, and derivatives thereof, as well as production ofsprouted seedlings systems.

Clonal Plants

Clonal roots maintain RNA viral expression vectors and stably producetarget protein uniformly in an entire root over extended periods of timeand multiple subcultures. In contrast to plants, where a target gene iseliminated via recombination during cell-to-cell or long distancemovement, in root cultures the integrity of a viral vector is maintainedand levels of target protein produced over time are similar to thoseobserved during initial screening. Clonal roots allow for ease ofproduction of heterologous protein material for oral formulation ofantigen and vaccine compositions. Methods and reagents for generating avariety of clonal entities derived from plants which are useful forproduction of antigen (e.g., antigen proteins in accordance with theinvention) have been described previously and are known in the art (see,for example, PCT Publication WO 05/81905, which is incorporated hereinby reference). Clonal entities include clonal root lines, clonal rootcell lines, clonal plant cell lines, and clonal plants capable ofproduction of antigen (e.g., antigen proteins in accordance with theinvention). The invention further provides methods and reagents forexpression of antigen polynucleotide and polypeptide products in clonalcell lines derived from various plant tissues (e.g., roots, leaves), andin whole plants derived from single cells (clonal plants). Such methodsare typically based on use of plant viral vectors of various types.

For example, in one aspect, the invention provides methods of obtaininga clonal root line that expresses a polynucleotide encoding a Y. pestisantigen in accordance with the invention comprising steps of: (i)introducing a viral vector that comprises a polynucleotide encoding a Y.pestis antigen into a plant or portion thereof; and (it) generating oneor more clonal root lines from a plant. Clonal root lines may begenerated, for example, by infecting a plant or plant portion (e.g., aharvested piece of leaf) with an Agrobacterium (e.g., A. rhizogenes)that causes formation of hairy roots. Clonal root lines can be screenedin various ways to identify lines that maintain virus, lines thatexpress a polynucleotide encoding a Y. pestis antigen at high levels,etc. The invention further provides clonal root lines, e.g., clonal rootlines produced in accordance with the invention and further encompassesmethods of expressing polynucleotides and producing polypeptide(s)encoding Y. pestis antigen(s) using clonal root lines.

The invention further provides methods of generating a clonal root cellline that expresses a polynucleotide encoding a Y. pestis antigen inaccordance with the invention comprising steps of: (i) generating aclonal root line, cells of which contain a viral vector whose genomecomprises a polynucleotide encoding a Y. pestis antigen; (ii) releasingindividual cells from a clonal root line; and (iii) maintaining cellsunder conditions suitable for root cell proliferation. The inventionprovides clonal root cell lines and methods of expressingpolynucleotides and producing polypeptides using clonal root cell lines.

In one aspect, the invention provides methods of generating a clonalplant cell line that expresses a polynucleotide encoding a Y. pestisantigen in accordance with the invention comprising steps of: (i)generating a clonal root line, cells of which contain a viral vectorwhose genome comprises a polynucleotide encoding a Y. pestis antigen;(ii) releasing individual cells from a clonal root line; and (iii)maintaining cells in culture under conditions appropriate for plant cellproliferation. The invention further provides methods of generating aclonal plant cell line that expresses a polynucleotide encoding a Y.pestis antigen comprising steps of: (i) introducing a viral vector thatcomprises a polynucleotide encoding a Y. pestis antigen into cells of aplant cell line maintained in culture; and (ii) enriching for cells thatcontain viral vector. Enrichment may be performed, for example, by (i)removing a portion of cells from culture; (ii) diluting removed cells soas to reduce cell concentration; (iii) allowing diluted cells toproliferate; and (iv) screening for cells that contain viral vector.Clonal plant cell lines may be used for production of a Y. pestisantigen in accordance with the present invention.

The invention includes a number of methods for generating clonal plants,cells of which contain a viral vector that comprises a polynucleotideencoding Y. pestis antigen in accordance with the invention. Forexample, the invention provides methods of generating a clonal plantthat expresses a polynucleotide encoding Y. pestis antigen comprisingsteps of: (i) generating a clonal root line, cells of which contain aviral vector whose genome comprises a polynucleotide encoding Y. pestisantigen; (ii) releasing individual cells from a clonal root line; and(iii) maintaining released cells under conditions appropriate forformation of a plant. The invention further provides methods ofgenerating a clonal plant that expresses a polynucleotide encoding Y.pestis antigen comprising steps of: (i) generating a clonal plant cellline, cells of which contain a viral vector whose genome comprises apolynucleotide encoding a Y. pestis antigen; and (ii) maintaining cellsunder conditions appropriate for formation of a plant. In general,clonal plants according to the invention can express any polynucleotideencoding a Y. pestis antigen. Such clonal plants can be used forproduction of an antigen polypeptide.

As noted above, the present invention provides systems for expressing apolynucleotide or polynucleotide(s) encoding Y. pestis antigen(s) inaccordance with the invention in clonal root lines, clonal root celllines, clonal plant cell lines (e.g., cell lines derived from leaf,stem, etc.), and in clonal plants. A polynucleotide encoding a Y. pestisantigen is introduced into all ancestral plant cell using a plant viralvector whose genome includes polynucleotide encoding a Y. pestis antigenoperably linked to (i.e., under control of) a promoter. A clonal rootline or clonal plant cell line is established from a cell containingvirus according to ally of several techniques further described below. Aplant virus vector or portions thereof can be introduced into a plantcell by infection, by inoculation with a viral transcript or infectiouscDNA clone, by electroporation, by T-DNA mediated gene transfer, etc.

The following sections describe methods for generating clonal rootlines, clonal root cell lines, clonal plant cell lines, and clonalplants that express a polynucleotide encoding a Y. pestis antigen inaccordance with the invention are then described. A “root line” isdistinguished from a “root cell line” in that a root line producesactual root-like structures or roots while a root cell line consists ofroot cells that do not form root-like structures. Use of the term “line”is intended to indicate that cells of a line can proliferate and passgenetic information on to progeny cells. Cells of a cell line typicallyproliferate in culture without being part of an organized structure suchas those found in an intact plant. Use of the term “root line” isintended to indicate that cells in a root structure can proliferatewithout being part of a complete plant. It is noted that the term “plantcell” encompasses root cells. However, to distinguish methods inaccordance with the invention for generating root lines and root celllines from those used to directly generate plant cell lines fromnon-root tissue (as opposed to generating clonal plant cell lines fromclonal root lines or clonal plants derived from clonal root lines), theterms “plant cell” and “plant cell line” as used herein generally referto cells and cell lines that consist of non-root plant tissue. Plantcells can be, for example, leaf, stem, shoot, flower part, etc. It isnoted that seeds can be derived from clonal plants generated as derivedherein. Such seeds may contain viral vector as will plants obtained fromsuch seeds. Methods for obtaining seed stocks are well known in the art(see, for example, U.S Patent Publication 2004/093643; incorporatedherein by reference).

Clonal Root Lines

The present invention provides systems for generating a clonal root linein which a plant viral vector is used to direct expression of apolynucleotide encoding a Y. pestis antigen in accordance with theinvention. One or more viral expression vector(s) including apolynucleotide encoding a Y. pestis antigen operably linked to apromoter is introduced into a plant or a portion thereof according toany of a variety of known methods. For example, plant leaves can beinoculated with viral transcripts. Vectors themselves may be directlyapplied to plants (e.g., via abrasive inoculations, mechanized sprayinoculations, vacuum infiltration, particle bombardment, orelectroporation). Alternatively or additionally, virions may be prepared(e.g., from already infected plants), and may be applied to other plantsaccording to known techniques.

Where infection is to be accomplished by direct application of a viralgenome to a plant, any available technique may be used to prepare viralgenome. For example, many viruses that are usefully employed inaccordance with the present invention have ssRNA genomes, ssRNA may beprepared by transcription of a DNA copy of the genome, or by replicationof an RNA copy, either in vivo or in vitro. Given the readily available,easy-to-use in vitro transcription systems (e.g., SP6, T7, reticulocytelysate, etc.), and also the convenience of maintaining a DNA copy of anRNA vector, it is expected that ssRNA vectors will often be prepared byin vitro transcription, particularly with T7 or SP6 polymerase.Infectious cDNA clones can be used. Agrobacterially-mediated genetransfer can be used to transfer viral nucleic acids such as viralvectors (either entire viral genomes or portions thereof) to plant cellsusing, e.g., agroinfiltration, according to methods known in the art.

A plant or plant portion may then be then maintained (e.g., cultured orgrown) under conditions suitable for replication of viral transcript. Incertain embodiments in accordance with the invention virus spreadsbeyond tile initially inoculated cell, e.g., locally from cell to celland/or systemically from an initially inoculated leaf into additionalleaves. However, in some embodiments, virus does not spread. Thus viralvector may contain genes encoding functional MP and/or CP, but may belacking one or both of such genes. In general, viral vector isintroduced into (infects) multiple cells in the plant or portionthereof.

Following introduction of viral vector into a plant, leaves areharvested. In general, leaves may be harvested at any time followingintroduction of a viral vector.

However, it may be desirable to maintain a plant for a period of timefollowing introduction of a viral vector into a plant, e.g., a period oftime sufficient for viral replication and, optionally, spread of virusfrom cells into which it was initially introduced. A clonal root culture(or multiple cultures) is prepared, e.g., by known methods furtherdescribed below.

In general, any available method may be used to prepare a clonal rootculture from a plant or plant tissue into which a viral vector has beenintroduced. One such method employs genes that exist in certainbacterial plasmids. These plasmids are found in various species ofAgrobacterium that infect and transfer DNA to a wide variety oforganisms. As a genus, Agrobacteria can transfer DNA to a large anddiverse set of plant types including numerous dicot and monocotangiosperm species and gymnosperms (see, for example, Gelvin, 2003,Microbiol. Mol. Biol. Rev., 67:16, and references therein, all of whichare incorporated herein by reference). The molecular basis of genetictransformation of plant cells is transfer from bacterium and integrationinto plant nuclear genome of a region of a large tumor-inducing (Ti) orrhizogenic (Ri) plasmid that resides within various Agrobacterialspecies. This region is referred to as the T-region when present in theplasmid and as T-DNA when excised from plasmid. Generally, asingle-stranded T-DNA molecule is transferred to a plant cell innaturally occurring Agrobacterial infection and is ultimatelyincorporated (in double-stranded form) into the genome. Systems based onTi plasmids are widely used for introduction of foreign genetic materialinto plants and for production of transgenic plants.

Infection of plants with various Agrobacterial species and transfer ofT-DNA has a number of effects. For example, A. tumefaciens causes crowngall disease while A. rhizogenes causes development of hairy roots atthe site of infection, a condition known as “hairy root disease.” Eachroot arises from a single genetically transformed cell. Thus root cellsin roots are clonal, and each root represents a clonal population ofcells. Roots produced by A. rhizogenes infection are characterized by ahigh growth rate and genetic stability (Girt et al., 2000, Biotech.Adv., 18:1, and references therein, all of which are incorporated hereinby reference). In addition, such roots are able to regenerategenetically stable plants (Giri 2000, supra).

In general, the present invention encompasses use of any strain ofAgrobacteria, particularly A. rhizogenes, that is capable of inducingformation of roots from plant cells. As mentioned above, a portion ofthe Ri plasmid (Ri T-DNA) is responsible for causing hairy root disease.While transfer of this portion of the Ri plasmid to plant cells canconveniently be accomplished by infection with Agrobacteria harboringthe Ri plasmid, the invention encompasses use of alternative methods ofintroducing the relevant region into a plant cell. Such methods includeany available method of introducing genetic material into plant cellsincluding, but not limited to, biolistics, electroporation, PEG-mediatedDNA uptake, Ti-based vectors, etc. The relevant portions of Ri T-DNA canbe introduced into plant cells by use of a viral vector. Ri genes can beincluded in the same vector that contains a polynucleotide encoding a Y.pestis antigen in accordance with the invention or in a different viralvector, which can be the same or a different type to that of the vectorthat contains a polynucleotide encoding a Y. pestis antigen inaccordance with the invention. It is noted that the entire Ri T-DNA maynot be required for production of hairy roots, and the inventionencompasses use of portions of Ri T-DNA, provided that such portionscontain sufficient genetic material to induce root formation, as knownin the art. Additional genetic material, e.g., genes present within theRi plasmid but not within T-DNA, may be transferred to a plant cell inaccordance with the invention, particularly genes whose expressionproducts facilitate integration of T-DNA into plant cell DNA.

In order to prepare a clonal root line in accordance with certainembodiments, harvested leaf portions are contacted with A. rhizogenesunder conditions suitable for infection and transformation. Leafportions are maintained in culture to allow development of hairy roots.Each root is clonal, i.e., cells in the root are derived from a singleancestral cell into which Ri T-DNA was transferred. In accordance withthe invention, a portion of such ancestral cells will contain a viralvector. Thus cells in a root derived from such an ancestral cell maycontain viral vector since it will be replicated and will be transmittedduring cell division. Thus a high proportion (e.g. at least 50%, atleast 75%, at least 80%, at least 90%, at least 95%), all (i.e., 100%),or substantially all (e.g., at least 98%) of cells will contain viralvector. It is noted that since viral vector is inherited by daughtercells within a clonal root, movement of viral vector within the root isnot necessary to maintain viral vector throughout the root. Individualclonal hairy roots may be removed from the leaf portion and furthercultured. Such roots are also referred to herein as root lines. Isolatedclonal roots continue to grow following isolation.

A variety of different clonal root lines have been generated usingmethods in accordance with the invention. These root lines weregenerated using viral vectors containing polynucleotide(s) encoding a Y.pestis antigen in accordance with the invention (e.g., encodingincluding Y. pestis protein(s), portions, fragments, domains, variants,and/or fusions thereof). Root lines were tested by western blot. Rootlines displayed a variety of different expression levels of variouspolypeptides. Root lines displaying high expression were selected andfurther cultured. These root lines were subsequently tested again andshown to maintain high levels of expression over extended periods oftime, indicating stability. Expression levels were comparable to orgreater than expression in intact plants infected with the same viralvector used to generate clonal root lines. In addition, stability ofexpression of root lines was superior to that obtained in plantsinfected with the same viral vector. Up to 80% of such virus-infectedplants reverted to wild type after 2-3 passages. (Such passages involvedinoculating plants with transcripts, allowing infection (local orsystemic) to become established, taking a leaf sample, and inoculatingfresh plants that are subsequently tested for expression).

Root lines may be cultured on a large scale for production of antigen inaccordance with the invention polypeptides as discussed further below,it is noted that clonal root lines (and cell lines derived from clonalroot lines) can generally be maintained in medium that does not includevarious compounds, e.g., plant growth hormones such as auxins,cytokinins, etc., that are typically employed in culture of root andplant cells. This feature greatly reduces expense associated with tissueculture, and the inventors expect that it will contribute significantlyto economic feasibility of protein production using plants.

Any of a variety of methods may be used to select clonal roots thatexpress a polynucleotide encoding Y. pestis antigen(s) in accordancewith the invention. Western blots, ELISA assays, etc., can be used todetect an encoded polypeptide. In the case of detectable markers such asGFP, alternative methods such as visual screens can be performed. If aviral vector that contains a polynucleotide that encodes a selectablemarker is used, an appropriate selection can be imposed (e.g., leafmaterial and/or roots derived therefrom can be cultured in the presenceof an appropriate antibiotic or nutritional condition and survivingroots identified and isolated). Certain viral vectors contain two ormore polynucleotide(s) encoding Y. pestis antigen(s) in accordance withthe invention, e.g., two or more polynucleotides encoding differentpolypeptides. If one of these is a selectable or detectable marker,clonal roots that are selected or detected by selecting for or detectingexpression of a marker will have a high probability of also expressing asecond polynucleotide. Screening for root lines that contain particularpolynucleotides can also be performed using PCR and other nucleic aciddetection methods.

Alternatively or additionally, clonal root lines can be screened forpresence of virus by inoculating host plants that will form locallesions as a result of virus infection (e.g., hypersensitive hostplants). For example, 5 mg of root tissue can be homogenized in 50 μl ofphosphate buffer and used to inoculate a single leaf of a tobacco plant.If virus is present in root cultures, within two to three dayscharacteristic lesions will appear on infected leaves. This means thatroot line contains recombinant virus that carries a polynucleotideencoding a Y. pestis antigen in accordance with the invention (a targetgene). If no local lesions are formed, there is no virus, and the rootline is rejected as negative. This method is highly time- andcost-efficient. After initially screening for the presence of virus,roots that contain virus may be subjected to secondary screening, e.g.,by western blot or ELISA to select high expressers. Additional screens,e.g., screens for rapid growth, growth in particular media or underparticular environmental conditions, etc., can be applied. Thesescreening methods may, in general, be applied in the development of anyof clonal root lines, clonal root cell lines, clonal plant cell lines,and/or clonal plants described herein.

As will be evident to one of ordinary skill in the art, a variety ofmodifications may be made to the description of methods in accordancewith the invention for generating clonal root lines that contain a viralvector. Such modifications are within the scope of the invention. Forexample, while it is generally desirable to introduce viral vector intoan intact plant or portion thereof prior to introduction of Ri T-DNAgenes, in certain embodiments, Ri-DNA is introduced prior to introducingviral vector. In addition, it is possible to contact intact plants withA. rhizogenes rather than harvesting leaf portions and then exposingthem to bacterium.

Other methods of generating clonal mot lines from single cells of aplant or portion thereof that harbor a viral vector can be used (i.e.,methods not using A. rhizogenes or genetic material from the Riplasmid). For example, treatment with certain plant hormones orcombinations of plant hormones is known to result in generation of rootsfrom plant tissue.

Clonal Cell Lines Derived from Clonal Root Lines

As described above, the invention provides methods for generating clonalmot lines, wherein cells in root lines contain a viral vector. As iswell known in the art, a variety of different cell lines can begenerated from roots. For example, root cell lines can be generated fromindividual root cells obtained from a root using a variety of knownmethods. Such root cell lines may be obtained from various differentroot cell types within a root. In general, root material is harvestedand dissociated (e.g., physically and/or enzymatically digested) torelease individual root cells, which are then further cultured. Completeprotoplast formation is generally not necessary. If desired, root cellscan be plated at very dilute cell concentrations, so as to obtain rootcell lines from single root cells. Root cell lines derived in thismanner are clonal root cell lines containing viral vector. Such rootcell lines therefore exhibit stable expression of a polynucleotideencoding a Y. pestis antigen in accordance with the invention. Clonalplant cell lines can be obtained in a similar manner from clonal roots,e.g., by culturing dissociated root cells in the presence of appropriateplant hormones. Screens and successive rounds of enrichment can be usedto identify cell lines that express a polynucleotide encoding a Y.pestis antigen at high levels. However, if the clonal root line fromwhich a call line is derived already expresses at high levels, suchadditional screens may be unnecessary.

As in the case of clonal root lines, cells of a clonal root cell lineare derived from a single ancestral cell that contains viral vector andwill, therefore, also contain viral vector since it will be replicatedmad will be transmitted during cell division. Thus a high proportion(e.g. at least 50%, at least 75%, at least 80%, at least 90%, at least95%), all (i.e., 100%), or substantially all (e.g., at least 98%) ofcells will contain viral vector. It is noted that since viral vector isinherited by daughter cells within a clonal root cell line, movement ofviral vector among cells is not necessary to maintain viral vector.Clonal root cell lines can be used for production of a polynucleotideencoding Y. pestis antigen as described below.

Clonal Plant Cell Lines

The present invention provides methods for generating a clonal plantcell line in which a plant viral vector is used to direct expression ofa polynucleotide encoding a Y. pestis antigen in accordance with theinvention. According to methods in accordance with the invention, one ormore viral expression vector(s) including a polynucleotide encoding a Y.pestis antigen operably linked to a promoter is introduced into cells ofa plant cell line that is maintained in cell culture. A number of plantcell lines from various plant types are known in the art, any of whichcan be used. Newly derived cell lines can be generated according toknown methods for use in practicing the invention. A viral vector isintroduced into cells of a plant cell line according to any of a numberof methods. For example, protoplasts can be made and viral transcriptsthen electroporated into cells. Other methods of introducing a plantviral vector into cells of a plant cell line can be used.

A method for generating clonal plant cell lines in accordance with theinvention and a viral vector suitable for introduction into plant cells(e.g., protoplasts) can be used as follows: Following introduction ofviral vector, a plant cell line may be maintained in tissue culture.During this time viral vector may replicate, and polynucleotide(s)encoding a Y. pestis antigen(s) may be expressed. Clonal plant celllines are derived from culture, e.g., by a process of successiveenrichment. For example, samples may be removed from culture, optionallywith dilution so that the concentration of cells is low, and plated inPetri dishes in individual droplets. Droplets are then maintained toallow cell division.

It will be appreciated that droplets may contain a variable number ofcells, depending on initial density of the culture and amount ofdilution. Cells can be diluted such that most droplets contain either 0or 1 cell if it is desired to obtain clonal cell lines expressing apolynucleotide encoding a Y. pestis antigen after only a single round ofenrichment. However, it can be more efficient to select a concentrationsuch that multiple cells are present in each droplet and then screendroplets to identify those that contain expressing cells. In general,any appropriate screening procedure can be employed. For example,selection or detection of a detectable marker such as GFP can be used.Western blots or ELISA assays can be used. Individual droplets (100 μl)contain more than enough cells for performance of these assays. Multiplerounds of enrichment are performed to isolate successively higherexpressing cell lines. Single clonal plant cell lines (i.e., populationsderived from a single ancestral cell) can be generated by furtherlimiting dilution using standard methods for single cell cloning.However, it is not necessary to isolate individual clonal lines. Apopulation containing multiple clonal cell lines can be used forexpression of a polynucleotide encoding one or more Y. pestisantigen(s).

In general, certain considerations described above for generation ofclonal root lines apply to generation of clonal plant cell lines. Forexample, a diversity of viral vectors containing one or morepolynucleotide(s) encoding a Y. pestis antigen(s) in accordance with theinvention can be used as combinations of multiple different vectors.Similar screening methods can be used. As in the case of clonal rootlines and clonal root cell lines, cells of a clonal plant cell line arederived from a single ancestral cell that contains viral vector andwill, therefore, also contain viral vector since it will be replicatedand will be transmitted during cell division. Thus a high proportion(e.g. at least 50%, at least 75%, at least 80%, at least 90%, at least95%), all (i.e., 100%), or substantially all (e.g., at least 98%) ofcells will contain viral vector. It is noted that since viral vector isinherited by daughter cells within a clonal plant cell line, movement ofviral vector among cells is not necessary to maintain viral vector. Aclonal plant cell line can be used for production of a polypeptideencoding a Y. pestis antigen as described below.

Clonal Plants

Clonal plants can be generated from clonal roots, clonal root celllines, and/or clonal plant cell lines produced according to variousmethods described above. Methods for generation of plants from roots,root cell lines, and plant cell lines such as clonal root lines, clonalroot cell lines, and clonal plant cell lines described herein are wellknown in the art (see, e.g., Peres et al., 2001, Plant Cell, Tissue,Organ Culture, 65:37; and standard reference works on plant molecularbiology and biotechnology cited elsewhere herein). The inventiontherefore provides a method of generating a clonal plant comprisingsteps of (i) generating a clonal root line, clonal root cell line, orclonal plant cell line according to any of the methods described above;and (ii) generating a whole plant from a clonal root line, clonal rootcell line, or clonal plant. Clonal plants may be propagated and grownaccording to standard methods.

As in the case of clonal root lines, clonal root cell lines, and clonalplant cell lines, cells of a clonal plant are derived from a singleancestral cell that contains viral vector and will, therefore, alsocontain viral vector since it will be replicated and will be transmittedduring cell division. Thus a high proportion (e.g. at least 50%, atleast 75%, at least 80%, at least 90%, at least 95%), all (i.e., 100%),or substantially all (e.g., at least 98%) of cells will contain viralvector. It is noted that since viral vector is inherited by daughtercells within a clonal plant, movement of viral vector is not necessaryto maintain viral vector.

Sprouts and Sprouted Seedling Plant Expression Systems

Systems and reagents for generating a variety of sprouts and sproutedseedlings which are useful for production of Y. pestis antigen(s)according to the present invention have been described previously andare known in the art (see, for example, PCT Publication WO 04/43886;incorporated herein by reference). The present invention furtherprovides sprouted seedlings, which may be edible, as a biomasscontaining a Y. pestis antigen. In certain aspects, biomass is provideddirectly for consumption of antigen containing compositions. In someaspects, biomass is processed prior to consumption, for example, byhomogenizing, crushing, drying, or extracting. In certain aspects, Y.pestis antigen is purified from biomass and formulated into apharmaceutical composition.

Additionally provided are methods for producing Y. pestis antigen(s) insprouted seedlings that can be consumed or harvested live (e.g.,sprouts, sprouted seedlings of the Brassica genus). In certain aspects,the present invention involves growing a seed to an edible sproutedseedling in a contained, regulatable environment (e.g., indoors, in acontainer, etc.). A seed can be a genetically engineered seed thatcontains an expression cassette encoding a Y. pestis antigen, whichexpression is driven by an exogenously inducible promoter. A variety ofexogenously inducible promoters can be used that are inducible, forexample, by light, heat, phytohormones, nutrients, etc.

In related embodiments, the present invention provides methods ofproducing Y. pestis antigen(s) in sprouted seedlings by first generatinga seed stock for a sprouted seedling by transforming plants with anexpression cassette that encodes Y. pestis antigen using anAgrobacterium transformation system, wherein expression of a Y. pestisantigen is driven by an inducible promoter. Transgenic seeds can beobtained from a transformed plant, grown in a contained, regulatableenvironment, and induced to express a Y. pestis antigen.

In some embodiments methods are provided that involves infectingsprouted seedlings with a viral expression cassette encoding a Y. pestisantigen, expression of which may be driven by any of a viral promoter oran inducible promoter. Sprouted seedlings are grown for two to fourteendays in a contained, regulatable environment or at least untilsufficient levels of Y. pestis antigen have been obtained forconsumption or harvesting.

The present invention further provides systems for producing Y. pestisantigen(s) in sprouted seedlings that include a housing unit withclimate control and a sprouted seedling containing an expressioncassette that encodes one or more Y. pestis antigens, wherein expressionis driven by a constitutive or inducible promoter. Systems can provideunique advantages over an outdoor environment or greenhouse, whichcannot be controlled. Thus, the present invention enables a grower toprecisely time induction of expression of Y. pestis antigen. It cangreatly reduce time and cost of producing Y. pestis antigen(s).

In certain aspects, transiently transfected sprouts contain viral vectorsequences encoding a Y. pestis antigen in accordance with the invention.Seedlings are grown for a time period so as to allow for production ofviral nucleic acid in sprouts, followed by a period of growth whereinmultiple copies of virus are produced, thereby resulting in productionof Y. pestis antigen(s).

In certain aspects, genetically engineered seeds or embryos that containa nucleic acid encoding Y. pestis antigen(s) are grown to sproutedseedling stage in a contained, regulatable environment. A contained,regulatable environment may be a housing unit or room in which seeds canbe grown indoors. All environmental factors of a contained, regulatableenvironment may be controlled. Since sprouts do not require light togrow, and lighting can be expensive, genetically engineered seeds orembryos may be grown to sprouted seedling stage indoors in absence oflight.

Other environmental factors that can be regulated in a contained,regulatable environment in accordance with the invention includetemperature, humidity, water, nutrients, gas (e.g., O₂ or CO₂ content orair circulation), chemicals (small molecules such as sugars and sugarderivatives or hormones such as such as phytohormones gibberellic orabsisic acid, etc.) and the like.

According to certain methods, expression of a nucleic acid encoding a Y.pestis antigen may be controlled by an exogenously inducible promoter.Exogenously inducible promoters are caused to increase or decreaseexpression of a nucleic acid in response to an external, rather than aninternal stimulus. A number of environmental factors can act as inducersfor expression of nucleic acids carried by expression cassettes ofgenetically engineered sprouts. A promoter may be a heat-induciblepromoter, such as a heat-shock promoter. For example, using asheat-shock promoter, temperature of a contained environment may simplybe raised to induce expression of a nucleic acid. Other promotersinclude light inducible promoters. Light-inducible promoters can bemaintained as constitutive promoters if light in a contained regulatableenvironment is always on. Alternatively or additionally, expression of anucleic acid can be turned on at a particular time during development bysimply turning on a light. A promoter may be a chemically induciblepromoter is used to induce expression of a nucleic acid. According tothese embodiments, a chemical could simply be misted or sprayed ontoseed, embryo, or seedling to induce expression of nucleic acid. Sprayingand misting can be precisely controlled and directed onto target seed,embryo, or seedling to which it is intended. A contained environment isdevoid of wind or air currents, which could disperse chemical away fromintended target, so that the chemical stays on the target for which itwas intended.

According to the present invention, time of expression is induced can beselected to maximize expression of a Y. pestis antigen in sproutedseedling by the time of harvest. Inducing expression in an embryo at aparticular stage of growth, for example, inducing expression in anembryo at a particular number of days after germination, may result inmaximum synthesis of a Y. pestis antigen at the time of harvest. To givebut one example, in some situations, inducing expression from a promoter4 days after germination may result in more protein synthesis thaninducing expression from the promoter after 3 days or after 5 days.Those skilled in the art will appreciate that maximizing expression canbe achieved by routine experimentation. In certain methods, sproutedseedlings are harvested at about 1, about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, or about 14 days after germination.

In cases where an expression vector has a constitutive promoter insteadof an inducible promoter, sprouted seedling may be harvested at acertain time after transformation of sprouted seedling. For example, ifa sprouted seedling were vitally transformed at an early stage ofdevelopment, for example, at embryo stage, sprouted seedlings may beharvested at a time when expression is at its maximumpost-transformation, e.g., at about 1, about 2, about 3, about 4, about5, about 6, about 7, about 8, about 9, about 10, about 11, about 12,about 13, or about 14 days post-transformation. It could be that sproutsdevelop one, two, three or more mouths post-transformation, depending ongermination of seed.

Generally, once expression of Y. pestis antigen(s) begins, seeds,embryos, or sprouted seedlings are allowed to grow until sufficientlevels of Y. pestis antigen(s) are expressed. In certain aspects,sufficient levels are levels that would provide a therapeutic benefit toa patient if harvested biomass were eaten raw. Alternatively oradditionally, sufficient levels are levels from which Y. pestis antigencan be concentrated or purified from biomass and formulated into apharmaceutical composition that provides a therapeutic benefit to apatient upon administration. Typically, Y. pestis antigen is not aprotein expressed in sprouted seedling in nature. At any rate, Y. pestisantigen is typically expressed at concentrations above that which wouldbe present in a sprouted seedling in nature.

Once expression of Y. pestis antigen is induced, growth is allowed tocontinue until sprouted seedling stage, at which time sprouted seedlingsare harvested. Sprouted seedlings can be harvested live. Harvesting livesprouted seedlings has several advantages including minimal effort andbreakage. Sprouted seedlings in accordance with the invention may begrown hydroponically, making harvesting a simple matter of lifting asprouted seedling from its hydroponic solution. No soil is required forgrowth of sprouted seedlings, but may be provided if deemed necessary ordesirable by the skilled artisan. Because sprouts can be grown withoutsoil, no cleansing of sprouted seedling material is required at the timeof harvest. Being able to harvest a sprouted seedling directly from itshydroponic environment without washing or scrubbing minimizes breakageof harvested material. Breakage and wilting of plants induces apoptosis.During apoptosis, certain proteolytic enzymes become active, which candegrade pharmaceutical protein expressed in a sprouted seedling,resulting in decreased therapeutic activity of a protein.Apoptosis-induced proteolysis can significantly decrease yield ofprotein from mature plants. Using methods in accordance with theinvention, apoptosis may be avoided when no harvesting takes place untilthe moment proteins are extracted from a plant.

For example, live sprouts may be ground, crushed, or blended to producea slurry of sprouted seedling biomass, in a buffer containing proteaseinhibitors. Buffer may be maintained at about 4° C. In some aspects,sprouted seedling biomass is air-dried, spray dried, frozen, orfreeze-dried. As in mature plants, some of these methods, such asair-drying, may result in a loss of activity of pharmaceutical protein.However, because sprouted seedlings are very small and have a largesurface area to volume ratio, this is much less likely to occur. Thoseskilled in the art will appreciate that many techniques for harvestingbiomass that minimize proteolysis of expressed protein are available andcould be applied to the present invention.

In some embodiments, sprouted seedlings are edible. In certainembodiments, sprouted seedlings expressing sufficient levels of Y.pestis antigens are consumed upon harvesting (e.g., immediately afterharvest, within minimal period following harvest) so that absolutely noprocessing occurs before sprouted seedlings are consumed. In this way,any harvest-induced proteolytic breakdown of Y. pestis antigen beforeadministration of Y. pestis antigen to a patient in need of treatment isminimized. For example, sprouted seedlings that are ready to be consumedcan be delivered directly to a patient. Alternatively or additionally,genetically engineered seeds or embryos are delivered to a patient inneed of treatment and grown to sprouted seedling stage by a patient. Inone aspect, a supply of genetically engineered sprouted seedlings isprovided to a patient, or to a doctor who will be treating patients, sothat a continual stock of sprouted seedlings expressing certaindesirable Y. pestis antigens may be cultivated. This may be particularlyvaluable for populations in developing countries, where expensivepharmaceuticals are not affordable or deliverable. The ease with whichsprouted seedlings in accordance with the invention can be grown makessprouted seedlings in accordance with the invention particularlydesirable for such developing populations.

The regulatable nature of a contained environment imparts advantages tothe present invention over growing plants in an outdoor environment. Ingeneral, growing genetically engineered sprouted seedlings that expresspharmaceutical proteins in plants provides a pharmaceutical productfaster (e.g., because plants are harvested younger) and with lesseffort, risk, and regulatory considerations than growing geneticallyengineered plants. A contained, regulatable environment used in thepresent invention reduces or eliminates risk of cross-pollinating plantsin nature.

For example, a heat inducible promoter likely would not be used outdoorsbecause outdoor temperature cannot be controlled. A promoter would beturned on any time that outdoor temperature rose above a certain level.Similarly, a promoter would be turned off every time outdoor temperaturedropped. Such temperature shifts could occur in a single day, forexample, turning expression on in the daytime and off at night. A heatinducible promoter, such as those described herein, would not even bepractical for use in a greenhouse, which is susceptible to climaticshifts to almost the same degree as outdoors. Growth of geneticallyengineered plants in a greenhouse is quite costly. In contrast, in thepresent system, every variable can be controlled so that a maximumamount of expression can be achieved with every harvest.

In certain embodiments, sprouted seedlings in accordance with theinvention are grown in trays that can be watered, sprayed, or misted atany time during development of sprouted seedling. For example, a traymay be fitted with one or more watering, spraying, misting, and drainingapparatus that can deliver and/or remove water, nutrients, chemicalsetc. at specific time and at precise quantities during development of asprouted seedling. For example, seeds require sufficient moisture tokeep them damp. Excess moisture drains through holes in trays intodrains in the floor of a room. Typically, drainage water is treated asappropriate for removal of harmful chemicals before discharge back intothe environment.

Another advantage of trays is that they can be contained within a verysmall space. Since no light is required for sprouted seedlings to grow,trays containing seeds, embryos, or sprouted seedlings may be tightlystacked vertically on top of one another, providing a large quantity ofbiomass per unit floor space in a housing facility constructedspecifically for these purposes. In addition, stacks of trays can bearranged in horizontal rows within a housing unit. Once seedlings havegrown to a stage appropriate for harvest (about two to fourteen days)individual seedling trays are moved into a processing facility, eithermanually or by automatic means, such as a conveyor belt.

Systems in accordance with the invention are unique in that they providea sprouted seedling biomass, which is a source of a Y. pestisantigen(s). Whether consumed directly or processed into a form of apharmaceutical composition, because sprouted seedlings are grown in acontained, regulatable environment, sprouted seedling biomass and/orpharmaceutical composition derived from biomass can be provided to aconsumer at low cost. In addition, the fact that conditions for growthof sprouted seedlings can be controlled makes quality and purity ofproduct consistent. A contained, regulatable environment obviates manysafety regulations of the EPA that can prevent scientists from growinggenetically engineered agricultural products out of doors.

Transformed Sprouts

A variety of methods can be used to transform plant cells and producegenetically engineered sprouted seedlings. Two available methods fortransformation of plants that require that transgenic plant cell linesbe generated in vitro, followed by regeneration of cell lines into wholeplants include Agrobacterium tumefaciens mediated gene transfer andmicroprojectile bombardment or electroporation. Viral transformation isa more rapid and less costly method of transforming embryos and sproutedseedlings that can be harvested without an experimental or generationallag prior to obtaining desired product. For any of these techniques, theskilled artisan would appreciate how to adjust and optimizetransformation protocols that have traditionally been used for plants,seeds, embryos, or spouted seedlings.

Agrobacterium Transformation Expression Cassettes

Agrobacterium is a representative genus of the gram-negative familyRhizobiaceae. This species is responsible for plant tumors such as crowngall and hairy root disease. In dedifferentiated plant tissue, which ischaracteristic of tumors, amino acid derivatives known as opines areproduced by the Agrobacterium and catabolized by the plant. Bacterialgenes responsible for expression of opines are a convenient source ofcontrol elements for chimeric expression cassettes. According to thepresent invention, Agrobacterium transformation system may be used togenerate edible sprouted seedlings, which are merely harvested earlierthan mature plants. Agrobacterium transformation methods can easily beapplied to regenerate sprouted seedlings expressing Y. pestis antigens.

In general, transforming plants involves transformation of plant cellsgrown in tissue culture by co-cultivation with an Agrobacteriumtumefaciens carrying a plant/bacterial vector. The vector contains agene encoding a Y. pestis antigen. An Agrobacterium transfers vector toplant host cell and is then eliminated using antibiotic treatment.Transformed plant cells expressing Y. pestis antigen are selected,differentiated, and finally regenerated into complete plantlets (Hellenset al., 2000, Plant Mol. Biol., 42:819; Pilon-Smits et al., 1999, PlantPhysiolog., 119:123; Barfield et al., 1991, Plant Cell Reports, 10:308;and Riva et al., 1998, J. Biotech., 1 (3); each of which is incorporatedby reference herein).

Expression vectors for use in the present invention include a gene (orexpression cassette) encoding a Y. pestis antigen designed for operationin plants, with companion sequences upstream and downstream of anexpression cassette. Companion sequences are generally of plasmid orviral origin and provide necessary characteristics to a vector totransfer DNA from bacteria to the desired plant host.

A basic bacterial/plant vector construct may desirably provide a broadhost range prokaryote replication origin, a prokaryote selectablemarker. Suitable prokaryotic selectable markers include resistancetoward antibiotics such as ampicillin or tetracycline. Other DNAsequences encoding additional functions that are well known in the artmay be present in a vector.

Agrobacterium T-DNA sequences are required for Agrobacterium mediatedtransfer of DNA to a plant chromosome. Tumor-inducing genes of T-DNA aretypically removed and replaced with sequences encoding a Y. pestisantigen. T-DNA border sequences are retained because they initiateintegration of T-DNA region into a plant genome. If expression of Y.pestis antigen is not readily amenable to detection, a bacterial/plantvector construct may include a selectable marker gene suitable fordetermining if a plant cell has been trans formed, e.g., nptII kanamycinresistance gene. On the same or different bacterial/plant vector (Tiplasmid) are Ti sequences. Ti sequences include virulence genes, whichencode a set of proteins responsible for excision, transfer andintegration of T-DNA into a plant genome (Schell, 1987, Science,237:1176; incorporated herein by reference). Other sequences suitablefor permitting integration of heterologous sequence into a plant genomemay include transposon sequences, and the like, for homologousrecombination.

Certain constructs will include an expression cassette encoding anantigen protein. One, two, or more expression cassettes may be used in agiven transformation. A recombinant expression cassette contains, inaddition to a Y. pestis antigen encoding sequence, at least thefollowing elements: a promoter region, plant 5′ untranslated sequences,initiation codon (depending upon whether or not an expressed gene hasits own), and transcription and translation termination sequences, inaddition, transcription and translation terminators may be included inexpression cassettes or chimeric genes in accordance with the invention.Signal secretion sequences that allow processing and translocation of aprotein, as appropriate, may be included in an expression cassette. Avariety of promoters, signal sequences, and transcription andtranslation terminators are described, for example, in Lawton et al.(1987, Plant Mol. Biol., 9:315; incorporated herein by reference) and inU.S. Pat. No. 5,888,789 (incorporated herein by reference), in addition,structural genes for antibiotic resistance are commonly utilized as aselection factor (Fraley et al. 1983, Proc. Natl. Acad. Sci., USA,80:4803, incorporated herein by reference). Unique restriction enzymesites at the 5′ and 3′ ends of a cassette allow for easy insertion intoa pre-existing vector. Other binary vector systems forAgrobacterium-mediated transformation, carrying at least one T-DNAborder sequence are described (PCT/EP99/07414, incorporated herein byreference).

Regeneration

Seeds of transformed plants may be harvested, dried, cleaned, and testedfor viability and for presence and expression of a desired gene product.Once this has been determined, seed stock is typically stored underappropriate conditions of temperature, humidity, sanitation, andsecurity to be used when necessary. Whole plants may then be regeneratedfrom cultured protoplasts, e.g., as described in Evans et al. (Handbookof Plant Cell Cultures, Vol. 1, MacMillan Publishing Co., New York,N.Y., 1983, incorporated herein by reference); and in Vasil (ed., CellCulture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Fla.,Vol. I, 1984, and Vol. III, 1986, incorporated herein by reference). Incertain aspects, plants are regenerated only to sprouted seedling stage.In some aspects, whole plants are regenerated to produce seed stocks andsprouted seedlings are generated from seeds of a seed stock.

All plants from which protoplasts can be isolated and cultured to givewhole, regenerated plants can be transformed by the present invention sothat whole plants are recovered that contain a transferred gene. It isknown that practically all plants can be regenerated from cultured cellsor tissues, including, but not limited to, all major species of plantsthat produce edible sprouts. Some suitable plants include alfalfa, mungbean, radish, wheat, mustard, spinach, carrot, beet, onion, garlic,celery, rhubarb, a leafy plant such as cabbage or lettuce, watercress orcress, herbs such as parsley, mint, or clovers, cauliflower, broccoli,soybean, lentils, edible flowers such as sunflower, etc.

Means for regeneration vary from one species of plants to the next.However, those skilled in the art will appreciate that generally asuspension of transformed protoplasts containing copies of aheterologous gene is first provided. Callus tissue is formed and shootsmay be induced from callus and subsequently rooted. Alternatively oradditionally, embryo formation can be induced from a protoplastsuspension. These embryos germinate as natural embryos to form plants.Steeping seed in water or spraying seed with water to increase themoisture content of a seed to between 35%-45% initiates germination. Forgermination to proceed, seeds are typically maintained in air saturatedwith water under controlled temperature and airflow conditions. Culturemedia generally contains various amino acids and hormones, such as auxinand cytokinins. In some embodiments, it is advantageous to add glutamicacid and proline to the medium, especially for such species as alfalfa.Shoots and roots normally develop simultaneously. Efficient regenerationtypically depends on the medium, the genotype, and the history of theculture. If these three variables are controlled, then regeneration canbe fully reproducible and repeatable.

Mature plants, grown from transformed plant cells, are selfed andnon-segregating, homozygous transgenic plants are identified. An inbredplant produces seeds containing antigen-encoding sequences in accordancewith the invention. Such seeds can be germinated and grown to sproutedseedling stage to produce Y. pestis antigen(s) according to the presentinvention. In related embodiments, seeds may be formed into seedproducts and sold with instructions on how to grow seedlings to anappropriate sprouted seedling stage for administration or harvestinginto a pharmaceutical composition. In some related embodiments, hybridsor novel varieties embodying desired traits may be developed from inbredplants in accordance with the invention.

Direct Integration

Direct integration of DNA fragments into the genome of plant cells bymicroprojectile bombardment or electroporation may be used in thepresent invention (see, e.g., Kikkert, et al., 1999, Plant: J. Tiss.Cult. Assoc., 35:43; and Bates, 1994, Mol. Biotech., 2:135; both ofwhich are incorporated herein by reference). More particularly, vectorsthat express Y. pestis antigen(s) can be introduced into plant cells bya variety of techniques. As described above, vectors may includeselectable markers for use in plant cells. Vectors may include sequencesthat allow their selection and propagation in a secondary host, such assequences containing an origin of replication and selectable marker.Typically, secondary hosts include bacteria and yeast. In someembodiments, a secondary host is bacteria (e.g., Escherichia coli, theorigin of replication is a colE1-type origin of replication) and aselectable marker is a gene encoding ampicillin resistance. Suchsequences are well known in the art and are commercially available(e.g., Clontech, Palo Alto, Calif. or Stratagene, La Jolla, Calif.).

Vectors in accordance with the invention may be modified to intermediateplant transformation plasmids that contain a region of homology to anAgrobacterium tumefaciens vector, a T-DNA border region fromAgrobacterium tumefaciens, and antigen encoding nucleic acids orexpression cassettes described above. Further vectors may include adisarmed plant tumor inducing plasmid of Agrobacterium tumefaciens.

According to this embodiment, direct transformation of vectors inventionmay involve microinjecting vectors directly into plant cells by use ofmicropipettes to mechanically transfer recombinant DNA (see, e.g.,Crossway, 1985, Mol. Gen. Genet., 202:179, incorporated herein byreference). Genetic material may be transferred into a plant cell usingpolyethylene glycols (see, e.g., Krens et al., 1982, Nature 296:72;incorporated herein by reference). Another method of introducing nucleicacids into plants via high velocity ballistic penetration by smallparticles with a nucleic acid either within the matrix of small beads orparticles, or on the surface (see, e.g., Klein et al., 1987, Nature327:70; and Knudsen et al., Planta, 185:330; both of which areincorporated herein by reference). Yet another method of introduction isfusion of protoplasts with other entities, either minicells, cells,lysosomes, or other fusible lipid-surfaced bodies (see, e.g., Fraley etal., 1982, Proc. Natl. Acad. Sci., USA, 79:1859; incorporated herein byreference). Vectors in accordance with the invention may be introducedinto plant cells by electroporation (see, e.g., Fromm et al. 1985, Proc.Natl. Acad. Sci., USA, 82:5824; incorporated herein by reference).According to this technique, plant protoplasts are electroporated in thepresence of plasmids containing a gene construct. Electrical impulses ofhigh field strength reversibly permeabilize biomembranes allowingintroduction of plasmids. Electroporated plant protoplasts reform thecell wall divide and form plant callus, which can be regenerated to formsprouted seedlings in accordance with the invention. Those skilled inthe art will appreciate how to utilize these methods to transform plantscells that can be used to generate edible sprouted seedlings.

Viral Transformation

Similar to conventional expression systems, plant viral vectors can beused to produce full-length proteins, including full length antigen.According to the present invention, plant virus vectors may be used toinfect and produce antigen(s) in seeds, embryos, sprouted seedlings,etc. Viral system that can be used to express everything from shortpeptides to large complex proteins. Specifically, using tobamoviralvectors is described, for example, by McCormick et al. (1999, Proc.Natl. Acad. Sci., USA, 96:703; Kumagai et al. 2000, Gene, 245:169; andVerch et al., 1998, J. Immunol. Methods, 220:69; all of which areincorporated herein by reference). Thus, plant viral vectors have ademonstrated ability to express short peptides as well as large complexproteins.

In certain embodiments, transgenic sprouts, which express Y. pestisantigen, are generated utilizing a host/virus system. Transgenic sproutsproduced by viral infection provide a source of transgenic protein thathas already been demonstrated to be safe. For example, sprouts are freeof contamination with animal pathogens. Unlike, for example, tobacco,proteins from an edible sprout could at least in theory be used in oralapplications without purification, thus significantly reducing costs. Inaddition, a virus/sprout system oilers a much simpler, less expensiveroute for scale-up and manufacturing, since transgenes are introducedinto virus, which can be grown up to a commercial scale within a fewdays. In contrast, transgenic plants can require up to 5-7 years beforesufficient seed or plant material is available for large-scale trials orcommercialization.

According to the present invention, plant RNA viruses have certainadvantages, which make them attractive as vectors for foreign proteinexpression. Molecular biology and pathology of a number of plant RNAviruses are well characterized and there is considerable knowledge ofvirus biology, genetics, and regulatory sequences. Most plant RNAviruses have small genomes and infectious cDNA clones are available tofacilitate genetic manipulation. Once infectious virus material enters asusceptible host cell, it replicates to high levels and spreads rapidlythroughout an entire sprouted seedling (one to fourteen dayspost-inoculation, e.g., about 1, about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, or about 14 days post-inoculation). Virus particles are easily andeconomically recovered from infected sprouted seedling tissue. Viruseshave a wide host range, enabling use of a single construct for infectionof several susceptible species. These characteristics are readilytransferable to sprouts.

Foreign sequences can be expressed from plant RNA viruses, typically byreplacing one of the viral genes with desired sequence, by insertingforeign sequences into a virus genome at an appropriate position, or byfusing foreign peptides to structural proteins of a virus. Moreover, anyof these approaches can be combined to express foreign sequences bytrans-complementation of vital functions of a virus. A number ofdifferent strategies exist as tools to express foreign sequences invirus-infected plants using tobacco mosaic virus (TMV), alfalfa mosaicvirus (A1MV), and chimeras thereof.

The genome of A1MV is a representative of the Bromoviridae family ofviruses and consists of three genomic RNAs (RNAs 1-3) and subgenomic RNA(RNA4). Genomic RNAs 1 and 2 encode virus replicase proteins P1 and P2,respectively. Genomic RNA3 encodes cell-to-cell movement protein P3 andcoat protein (CP). CP is translated from subgenomic RNA4, which issynthesized from genomic RNA3, and is required to start infection.Studies have demonstrated involvement of CP in multiple functions,including genome activation, replication, RNA stability, symptomformation, and RNA encapsidation (see e.g., Bol et al., 1971, Virology,46:73; Van Der Vossen et al., 1994, Virology 202:891; Yusibov et al.,Virology, 208:405; Yusibov et al., 1998, Virology, 242:1; Bol et al.,(Review, 100 refs.), 1999, J. Gen. Virol., 80:1089; De Graaff, 1995,Virology, 208:583; Jaspars et al., 1974, Adv. Virus Res., 19:37;Loesch-Fries, 1985, Virology, 146:177; Neeleman et al., 1991, Virology,181:687; Neeleman et al., 1993, Virology, 196: 883; Van Dar Kuyl et al.,1991, Virology, 183:731; and Van Dar Kuyl et al., 1991, Virology,185:496; all of which are incorporated herein by reference).

Encapsidation of viral particles is typically required for long distancemovement of virus from inoculated to un-inoculated parts of seed,embryo, or sprouted seedling and for systemic infection. According tothe present invention, inoculation can occur at any stage of plantdevelopment. In embryos and sprouts, spread of inoculated virus shouldbe very rapid. Virions of A1MV are encapsidated by a unique CP (24 kD),forming more than one type of particle. The size (30 to 60 nm in lengthand 18 nm in diameter) and shape (spherical, ellipsoidal, orbacilliform) of a particle depends on the size of an encapsidated RNA.Upon assembly, the N-terminus of A1MV CP is thought to be located on thesurface of virus particles and does not appear to interfere with virusassembly (Bol et al., 1971, Virology, 6:73; incorporated herein byreference). Additionally, ALMV CP with an additional 38-amino acidpeptide at its N-terminus forms particles in vitro and retainsbiological activity (Yusibov et al., 1995, J. Gen. Virol., 77:567;incorporated herein by reference).

A1MV has a wide host range, which includes a number of agriculturallyvaluable crop plants, including plant seeds, embryos, and sprouts.Together, these characteristics make ALMV CP an excellent candidate as acarrier molecule and AIMV an attractive candidate vector for expressionof foreign sequences in a plant at the sprout stage of development.Moreover, upon expression from a heterologous vector such as TMV, A1MVCP encapsidates TMV genome without interfering with virus infectivity(Yusibov et al., 1997, Proc. Natl. Acad. Sci., USA, 94:5784,incorporated herein by reference). This allows use of TMV as a carriervirus for A1MV CP fused to foreign sequences.

TMV, the prototype of tobamoviruses, has a genome consisting of a singleplus-sense RNA encapsidated with a 17.0 kD CP, which results inrod-shaped particles (about 300 nm in length). CP is the only structuralprotein of TMV and is required for encapsidation and long distancemovement of virus in an infected host (Saito et al., 1990, Virology176:329; incorporated herein by reference). 183 kid and 126 kD proteinsare translated from genomic RNA and are required for virus replication(Ishikawa et al., 1986, Nucleic Acids Res., 14:8291; incorporated hereinby reference). 30 kD protein is the cell-to-cell movement protein ofvirus (Meshi et al., 1987, EMBO J., 6:2557; incorporated herein byreference). Movement and coat proteins are translated from subgenomicmRNAs (Hunter et al., 1976, Nature, 260:759; Bruening et al., 1976,Virology, 71:498; and Beachy et al., 1976, Virology, 73:498, each ofwhich is incorporated herein by reference).

Other methods of transforming plant tissues include transforming aflower of a plant. Transformation of Arabidopsis thaliana can beachieved by dipping plant flowers into a solution of Agrobacteriumtumefaciens (Curtis et al., 2001, Transgenic Res., 10:363; and Qing etal., 2000, Molecular Breeding: New Strategies in Plant Improvement 1:67;both of which are incorporated herein by reference). Transformed plantsare formed in a population of seeds generated by “dipped” plants. At aspecific point during flower development, a pore exists in the ovarywall through which Agrobacterium tumefaciens gains access to theinterior of an ovary. Once inside the ovary, Agrobacterium tumefaciensproliferates and transforms individual ovules (Desfeux et al., 2000,Plant Physiology, 123:895; incorporated herein by reference).Transformed ovules follow the typical pathway of seed formation withinan ovary.

Agrobacterium-Mediated Transient Expression

As indicated herein, in many embodiments, systems for rapid (e.g.,transient) expression of proteins or polypeptides in plants aredesirable. Among other things, the present invention provides a powerfulsystem for achieving such rapid expression in plants that utilizes anagrobacterial construct to deliver a viral expression system encoding aprotein or polypeptide of interest. In some embodiments, any of the Y.pestis antigens described herein can be expressed utilizing launchvector technology, e.g., as described below, in some embodiments, launchvector constructs can also be utilized in the context of thermostableproteins, as described in more detail in the section entitled “Y. pestisPolypeptide Fusions with Thermostable Proteins.”

In some embodiments, according to the present invention, a “launchvector” is prepared that contains agrobacterial sequences includingreplication sequences and also contains plant viral sequences (includingself-replication sequences) that carry a gene encoding a protein orpolypeptide of interest. A launch vector is introduced into planttissue, typically by agroinfiltration, which allows substantiallysystemic delivery. For transient transformation, non-integrated T-DNAcopies of the launch vector remain transiently present in the nucleusand are transcribed leading to expression of the carrying genes (Kapilaet al., 1997, Plant Science, 122:101-108; incorporated herein byreference). Agrobacterium-mediated transient expression, differentlyfrom viral vectors, cannot lead to systemic spreading of expression of agene of interest. One advantage of this system is the possibility toclone genes larger than 2 kb to generate constructs that would beimpossible to obtain with viral vectors (Voinnet et al., 2003, Plant J.,33:949-56; incorporated herein by reference). Furthermore, using suchtechnique, it is possible to transform a plant with more than onetransgene, such that multimeric proteins (e.g., antibodies subunits ofcomplexed proteins) can be expressed and assembled. Furthermore, thepossibility of co-expression of multiple transgenes by means ofco-infiltration with different Agrobacterium can be taken advantage of,either by separate infiltration or using mixed cultures.

In certain embodiments, a launch vector includes sequences that allowfor selection (or at least detection) in Agrobacteria mad forselection/detection in infiltrated tissues. Furthermore, a launch vectortypically includes sequences that are transcribed in a plant to yieldviral RNA production, followed by generation of viral proteins.Furthermore, production of viral proteins and viral RNA yields rapidproduction of multiple copies of RNA encoding a pharmaceutically activeprotein of interest. Such production results in rapid protein productionof a target of interest in a relatively short period of time. Thus, ahighly efficient system for protein production can be generated.

Agroinfiltration utilizing viral expression vectors can be used toproduce limited quantities of protein of interest in order to verifyexpression levels before deciding if it is worth generating transgenicplants. Alternatively or additionally, agroinfiltration utilizing viralexpression vectors is useful for rapid generation of plants capable ofproducing huge amounts of protein as a primary production platform.Thus, this transient expression system can be used on industrial scale.

Further provided are any of a variety of different Agrobacterialplasmids, binary plasmids, or derivatives thereof such as pBIV, pBI1221,pGreen, etc., which can be used in these and other aspects of theinvention. Numerous suitable vectors are known in the art and can bedirected and/or modified according to methods known in the art, or thosedescribed herein so as to utilize in methods described provided herein.

One particular exemplary launch vector is pBID4. This vector containsthe 35S promoter of cauliflower mosaic virus (a DNA plant virus) thatdrives initial transcription of the recombinant viral genome followingintroduction into plants, and the nos terminator, the transcriptionalterminator of Agrobacterium nopaline synthase. The vector furthercontains sequences of the tobacco mosaic virus genome including genesfor vires replication (126/183K) and cell-t-cell movement (MP). Thevector further contains a gene encoding a polypeptide of interest,inserted into a unique cloning site within the tobacco mosaic viresgenome sequences and under the transcriptional control of the coatprotein subgenomic mRNA promoter. Because this “target gene” (i.e., geneencoding a protein or polypeptide of interest) replaces coding sequencesfor the TMV coat protein, the resultant viral vector is nakedself-replicating RNA that is less subject to recombination thanCP-containing vectors, and that cannot effectively spread and survive inthe environment. Left and right border sequences (LB and RB) delimit theregion of the launch vector that is transferred into plant cellsfollowing infiltration of plants with recombinant Agrobacterium carryingthe vector. Upon introduction of agrobacteria carrying this vector intoplant tissue (typically by agroinfiltration but alternatively byinjection or other means), multiple single-stranded DNA (ssDNA) copiesof sequence between LB and RB are generated and released in a matter ofminutes. These introduced sequences are then amplified by viralreplication. Translation of the target gene results in accumulation oflarge amounts of target protein or polypeptide in a short period oftime.

In some embodiments, Agrobacterium-mediated transient expressionproduces up to about 5 g or more of target protein per kg of planttissue. For example, in some embodiments, up to about 4, about 3, about2, about 1, or about 0.5 g of target protein is produced per kg of planttissue. In some embodiments, at least about 20-about 500 mg, or about50-about 500 of target protein, or about 50-about 200, or about 50,about 60, about 70, about 80, about 90, about 100, about 110, about 120,about 130, about 140, about 150, about 160, about 170, about 180, about190, about 200, about 250, about 300, about 350, about 400, about 450,about 500, about 550, about 600, about 650, about 700, about 750, about800, about 850, about 900, about 950, about 1000, about 1500, about1750, about 2000, about 2500, about 3000 rag, or more of protein per kgof plant tissue is produced.

In some embodiments, these expression levels are achieved within about6, about 5, about 4, about 3, or about 2 weeks from infiltration. Insome embodiments, these expression levels are achieved within about 10,about 9, about g, about 7, about 6, about 5, about 4, about 3, about 2days, or even 1 day, from introduction of an expression construct. Thus,the time from introduction (e.g., infiltration) to harvest is typicallyless than about 2 weeks, less than about 10 days, less than about 1week, or less than a few days. Furthermore, the invention allowsproduction of protein within about 8 weeks or less from the selection ofamino acid sequence (even including time for “preliminary” expressionstudies). Also, each batch of protein can typically be produced withinabout 8 weeks, about 6, weeks, about 5 weeks, or less. Those of ordinaryskill in the art will appreciate that these numbers may vary somewhatdepending on the type of plant used. Most sprouts, including peas, willfall within the numbers given. Nicotiana benthamiana, however, may begrown longer, particularly prior to infiltration, as they are slowergrowing (from a much smaller seed). Other expected adjustments will beclear to those of ordinary skill in the art based on biology of theparticular plants utilized. In some embodiments, certain pea varietiesincluding for example, marrowfat pea, bill jump pea, yellow trapper pea,speckled pea, and green pea are particularly useful.

The inventors have also found that various Nicotiana plants areparticularly useful in the practice of some aspects of the invention,including in particular Nicotiana benthamiana. In general, Nicotianabenthamiana plants are grown for a time sufficient to allow developmentof an appropriate amount of biomass prior to infiltration (i.e., todelivery of agrobacteria containing launch vector). Typically, plantsare grown for a period of more than about 3 weeks, more typically morethan about 4 weeks, or between about 5-about 6 weeks to accumulatebiomass prior to infiltration.

The present inventors have further surprisingly found that, althoughboth TMV and A1MV sequences can prove effective in such launch vectorconstructs, in some embodiments, AIMV sequences are particularlyefficient at ensuring high level production of delivered protein orpolypeptides.

Thus, in certain particular embodiments, proteins or polypeptides ofinterest are produced in plants (e.g., Nicotiana benthamiana) from alaunch vector that directs production of A1MV sequences carrying a geneof interest.

Yersinia pestis Polypeptide Fusions with Thermostable Proteins

In certain aspects, provided are Y. pestis antigen(s) comprising fusionpolypeptides which comprise a Y. pestis protein (or a fragment orvariant thereof) operably linked to a thermostable protein (e.g., LicB,LicKM, etc., and described in further detail below). Fusion polypeptidescan be produced in any available expression system known in the art(including, but not limited to, launch vector technology). In certainembodiments, fusion proteins are produced in a plant or portion thereof(e.g., plant, plant cell, root, sprout, etc.).

Enzymes or other proteins which are not found naturally in humans oranimal cells are particularly appropriate for use in fusion polypeptidesin accordance with the invention. Thermostable proteins that, whenfused, confer thermostability to a fusion product are useful.Thermostability allows produced protein to maintain conformation, andmaintain produced protein at room temperature. This feature facilitateseasy, time efficient and cost effective recovery of a fusionpolypeptide. A representative family of thermostable enzymes useful inaccordance with the invention is the glucanohydrolase family. Theseenzymes specifically cleave 1,4-β glucosidic bonds that are adjacent to1,3-13 linkages in mixed linked polysaccharides (Hahn et al., 1994 Proc.Natl. Acad. Sci., USA, 91:10417; incorporated herein by reference). Suchenzymes are found in cereals, such as oat and barley, and are also foundin a number of fungal and bacterial species, including C. thermocellum(Goldenkova et al., 2002, Mol. Biol. 36:698; incorporated herein byreference). Thus, desirable thermostable proteins for use in fusionpolypeptides in accordance with the invention include glycosidaseenzymes. Exemplary thermostable glycosidase proteins include thoserepresented by GenBank accession numbers selected from those set forthin Table 1, the contents of each of which are incorporated herein byreference by entire incorporation of the GenBank accession informationfor each referenced number. Exemplary thermostable enzymes of use infusion proteins in accordance with the invention include Clostridiumthermocellum P29716, Brevibacillus brevis P37073, and Rhodthermusmarinus P45798, each of which are incorporated herein by reference totheir GenBank accession numbers. Representative fusion proteinsillustrated in the Examples utilize modified thermostable enzymeisolated from Clostridium thermocellum, however, any thermostableprotein may be similarly utilized in accordance with the presentinvention.

TABLE 1 Thermostable Glycosidase Proteins P29716 (Beta-glucanaseClostridium thermocellum) P37073 (Beta-glucanase Brevibacillus brevis)1MVE_A (Beta-glucanase Fibrobacter sueeinogenes) P07883 (Extracellularagarase Streptomyces coelicolor) P23903 (Glucan endo-13-beta-glucosidaseA1 Bacillus circulans) P27051 (Beta-glucanase Bacillus licheniformis)P45797 (Beta-glucanase Paenibacillus polymyxa (Bacillus polymyxa))P37073 (Beta-glucanase Brevibacillus brevis) P45798 (Beta-glucanaseRhodothermus marinus) P38645 (Beta-glucosidase Thermobispora bispora)P40942 (Celloxylanase Clostridium stercorarium) P14002 (Beta-glucosidaseClostridium thermocellum) O33830 (Alpha-glucosidase Thermotoga maritima)O43097 (Xylanase Thermomyces lanuginosus) P54583 (Endo-glucanase E1Acidothermus cellulolyticus) P14288 (Beta-galactosidase Sulfolobusacidocaldarius) O52629 (Beta-galactosidase Pyrococcus woesei) P29094(Oligo-16-glucosidase Geobacillus thermoglucosidasius) P49067(Alpha-amylase Pyrococcus furiosus) JC7532 (Cellulase Bacillus species)Q60037 (Xylanase A Thermotoga maritima) P33558 (Xylanase A Clostridiumstercorarium) P04954 (Cellulase D Clostridium thermocellum) Q4J929(N-glycosylase Sulfolobus acidocaldarius) O33833 (Beta-fructosidaseThermotoga maritima) P49425 (Endo-14-beta-mannosidase Rhodothermusmarinus) P06279 (Alpha-amylase Geobacillus stearothermophilus) P45702(Xylanase Geobacillus stearothermophilus) P45703 P40943 P09961(Alpha-amylase 1 Dictyoglomus thermophilum) Q60042 (Xylanase AThermotoga neapolitana) AAN05438 (Beta-glycosidase Thermus thermophilus)AAN05439 AAN05437 (Sugar permease Thermus thermophilus) AAN05440(Beta-glycosidase Thermus filiformis) AAD43138 (Beta-glycosidaseThermosphaera aggregans)

When designing fusion proteins and polypeptides in accordance with theinvention, it is desirable, of course, to preserve immunogenicity of anantigen. Still further, it is desirable in certain aspects to provideconstructs which provide thermostability of a fusion protein. Thisfeature facilitates easy, time efficient and cost effective recovery ofa target antigen. In certain aspects, antigen fusion partners may beselected which provide additional advantages, including enhancement ofimmunogenicity, potential to incorporate multiple vaccine determinants,yet lack prior immunogenic exposure to vaccination subjects. Furtherbeneficial qualities of fusion peptides of interest include proteinswhich provide ease of manipulation for incorporation of one or moreantigens, as well as proteins which have potential to confer ease ofproduction, purification, and/or formulation for vaccine preparations.One of ordinary skill in the art will appreciate that three dimensionalpresentation can affect each of these beneficial characteristics.Preservation of immunity or preferential qualities therefore may affect,for example, choice of fusion partner and/or choice of fusion location(e.g., N-terminus, C-terminus, internal, combinations thereof).Alternatively or additionally, preferences may affects length of segmentselected for fusion, whether it be length of antigen, or length offusion partner selected.

The present inventors have demonstrated successful fusion of a varietyof antigens with a thermostable protein. For example, the presentinventors have used the thermostable carrier molecule LicB, alsoreferred to as lichenase, for production of fusion proteins. LicB is1,3-1,4-13 glucanase (LicB) from Clostridium thermocellum (GenBankaccession: X63355 [gi:40697]). LicB belongs to a family of globularproteins. Based on the three dimensional structure of LicB, its N- andC-termini are situated close to each other on the surface, in closeproximity to the active domain. LicB also has a loop structure exposedon the surface that is located far from the active domain. We havegenerated constructs such that the loop structure and N- and C-terminiof protein can be used as insertion sites for Y. pestis antigenpolypeptides. Y. pestis antigen polypeptides can be expressed as N- orC-terminal fusions or as inserts into the surface loop. Importantly,LicB maintains its enzymatic activity at low pH and at high temperature(up to about 75° C.). Thus, use of LicB as a carrier moleculecontributes advantages, including likely enhancement of target specificimmunogenicity, potential to incorporate multiple vaccine determinants,and straightforward formulation of vaccines that may be deliverednasally, orally or parenterally. Furthermore, production of LicB fusionsin plants should reduce the risk of contamination with animal or humanpathogens. See examples provided herein.

Fusion proteins comprising Y. pestis antigen may be produced in any of avariety of expression systems, including both in vitro and in vivosystems. One skilled in the art will readily appreciate thatoptimization of nucleic acid sequences for a particular expressionsystem is often desirable. For example, in the Exemplification providedherein, optimized sequence for expression of Y. pestis antigen-LicKMfusions in plants is provided (see Examples 1 and 2). Thus, any relevantnucleic acid encoding Y. pestis antigen(s) fusion protein(s) andfragments thereof in accordance with the invention is intended to beencompassed within nucleic acid constructs.

For production in plant systems, transgenic plants expressing Y. pestisantigen(s) (e.g., Y. pestis protein(s) or fragments or fusions thereof)may be utilized. Alternatively or additionally, transgenic plants may beproduced using methods well known in the art to generate stableproduction crops. Additionally, plants utilizing transient expressionsystems may be utilized for production of Y. pestis antigen(s). Whenutilizing plant expression systems, whether transgenic or transientexpression in plants is utilized, any of nuclear expression, chloroplastexpression, mitochondrial expression, or viral expression may be takenadvantage of according to the applicability of the system to antigendesired. Furthermore, additional expression systems for production ofantigens and fusion proteins in accordance with the present inventionmay be utilized. For example, mammalian expression systems (e.g.,mammalian cell lines (e.g., CHO, etc.)), bacterial expression systems(e.g., E. coli), insect expression systems (e.g., baculovirus), yeastexpression systems, and in vitro expression systems (e.g., reticulatelysates) may be used for expression of antigens and fusion proteins inaccordance with the invention.

Production and Isolation of Antigen

In general, standard methods known in the art may be used for culturingor growing plants, plant cells, and/or plant tissues in accordance withthe invention (e.g., clonal plants, clonal plant cells, clonal roots,clonal root lines, sprouts, sprouted seedlings, plants, etc.) forproduction of antigen(s). A wide variety of culture media andbioreactors have been employed to culture hairy root cells, root celllines, and plant cells (see, for example, Giri et al., 2000, Biotechnol.Adv., 18:1; Rao et al., 2002, Biotechnol. Adv., 20:101; and referencesin both of the foregoing, all of which are incorporated herein byreference). Clonal plants may be grown in any suitable manner.

In a certain embodiments, Y. pestis antigens in accordance with theinvention may be produced by any known method. In some embodiments, a Y.pestis antigen is expressed in a plant or portion thereof. Proteins areisolated and purified in accordance with conventional conditions andtechniques known in the art. These include methods such as extraction,precipitation, chromatography, affinity chromatography, electrophoresis,and the like. The present invention involves purification and affordablescaling up of production of Y. pestis antigen(s) using any of a varietyof plant expression systems known in the art and provided herein,including viral plant expression systems described herein.

In many embodiments, it will be desirable to isolate Y. pestisantigen(s) for vaccine products. Where a protein in accordance with theinvention is produced from plant tissue(s) or a portion thereof, e.g.,roots, root cells, plants, plant cells, that express them, methodsdescribed in further detail herein, or any applicable methods known inthe art may be used for any of partial or complete isolation from plantmaterial. Where it is desirable to isolate an expression product fromsome or all of plant cells or tissues that express it, any availablepurification techniques may be employed. Those of ordinary skill in theart are familiar with a wide range of fractionation and separationprocedures (see, for example, Scopes et al., Protein Purification:Principles and Practice, 3^(rd) Ed., Janson et al., 1993; ProteinPurification: Principles, High Resolution Methods, and Applications,Wiley-VCH, 1998; Springer-Verlag, NY, 1993; and Roe, ProteinPurification Techniques, Oxford University Press, 2001; each of which isincorporated herein by reference). Often, it will be desirable to rendera product more than about 50%, about 60%, about 70%, about 80%, about85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, or about 99% pure. See, e.g., U.S. Pat.Nos. 6,740,740 and 6,841,659 (each of which is incorporated herein byreference) for discussion of certain methods useful for purifyingsubstances from plant tissues or fluids.

Those skilled in the art will appreciate that a method Of obtainingdesired Y. Pestis antigen(s) product(s) is by extraction. Plant material(e.g., roots, leaves, etc.) may be extracted to remove desired productsfrom residual biomass, thereby increasing concentration and purity ofproduct. Plants may be extracted in a buffered solution. For example,plant material may be transferred into an amount of ice-cold water at aratio of one to one by weight that has been buffered with, e.g.,phosphate buffer. Protease inhibitors can be added as required. Plantmaterial can be disrupted by vigorous blending or grinding whilesuspended in buffer solution and extracted biomass removed by filtrationor centrifugation. Product carried in solution can be further purifiedby additional steps or converted to a dry powder by freeze-drying orprecipitation. Extraction can be carried out by pressing. Plants orroots can be extracted by pressing in a press or by being crushed asthey are passed through closely spaced rollers. Fluids expressed fromcrushed plants or roots are collected and processed according to methodswell known in the art. Extraction by pressing allows release of productsin a more concentrated form. However, overall yield of product may belower than if product were extracted in solution.

The present invention provides pharmaceutical antigen proteins fortherapeutic use, such as Y. pestis antigen(s) (e.g., Y. pestisprotein(s) or an immunogenic portion(s) thereof, or fusion proteinscomprising Y. pestis protein(s) or an immunogenic portion(s) thereof),active as agents for treatment and/or prophylaxis of Y. pestisinfection. Further, the invention provides vaccines for veterinary use,as Y. pestis antigen is active in veterinary applications. In certainembodiments, Y. pestis antigen(s) may be produced by plant(s) or portionthereof (e.g., root, cell, sprout, cell line, plant, etc.). In certainembodiments, provided Y. pestis antigens are expressed in plants, plantcells, and/or plant tissues (e.g., sprouts, sprouted seedlings, roots,root culture, clonal cells, clonal cell lines, clonal plants, etc.), andcan be used directly from plant or partially purified or purified inpreparation for pharmaceutical administration to a subject.

The present invention provides plants, plant cells, and plant tissuesexpressing Y. pestis antigen(s) that maintains pharmaceutical activitywhen administered to a subject in need thereof. Exemplary subjectsinclude vertebrates (e.g., mammals such as humans). According to thepresent invention, subjects include veterinary subjects such as bovines,ovines, canines, felines, etc. In certain aspects, an edible plant orportion thereof (e.g., sprout, root) is administered orally to a subjectin a therapeutically effective amount. In some aspects one or more Y.pestis antigen(s) is provided in a pharmaceutical preparation, asdescribed herein.

Vaccine compositions in accordance with the invention comprise one ormore Y. pestis antigens. In certain embodiments, at least two Y. pestisantigens are included in an administered vaccine composition.

According to the present invention, treatment of a subject with a Y.pestis antigen vaccine is intended to elicit a physiological effect. Avaccine protein may have healing curative or palliative propertiesagainst a disorder or disease and can be administered to amelioraterelieve, alleviate, delay onset of, reverse, and/or lessen symptoms orseverity of a disease or disorder. A vaccine comprising a Y. pestisantigen may have prophylactic properties and cml be used to prevent ordelay the onset of a disease or to lessen the severity of such disease,disorder, or pathological condition when it does emerge. A physiologicaleffect elicited by treatment of a subject with antigen according to thepresent invention can include an effective immune response such thatinfection by an organism is thwarted.

Pharmaceutical compositions in accordance with the invention can beadministered therapeutically or prophylactically. Compositions may beused to treat or prevent a disease. For example, any individual whosuffers from a disease (e.g. Yersinia pestis infection) or who is atrisk of developing a disease may be treated. It will be appreciated thatan individual can be considered at risk for developing a disease withouthaving been diagnosed with any symptoms of a disease (e.g. Yersiniapestis infection). For example, if an individual is known to have been,or to be intended to be, in situations with relatively high risk ofexposure to Y. pestis infection, that individual will be considered atrisk for developing the disease. Similarly, if members of anindividual's family or friends have been diagnosed with Y. pestisinfection, the individual may be considered to be at risk for developingthe disease. In some embodiments, if an individual has come into contactwith a non-human animal that has been diagnosed with Y. pestis infection(e.g., cat, dog, mouse, rat, horse, etc.), the individual may beconsidered to be at risk for developing the disease.

Administration

Yersinia pestis antigens in accordance with the invention and/orpharmaceutical compositions thereof (e.g., vaccines) may be administeredusing any amount and any route of administration effective fortreatment.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular composition, its mode ofadministration, its mode of activity, and the like. Y. pestis antigensare typically formulated in dosage unit form for ease of administrationand uniformity of dosage. It will be understood, however, that the totaldaily usage of the compositions of the present invention will be decidedby the attending physician within the scope of sound medical judgment.The specific therapeutically effective dose level for any particularsubject or organism will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific Y. pestis antigen employed; the specific pharmaceuticalcomposition administered; the half-life of the composition afteradministration; the age, body weight, general health, sex, and diet ofthe subject; the time of administration, route of administration, andrate of excretion of the specific compound employed; the duration of thetreatment; drags used in combination or coincidental with the specificcompound employed; and like factors, well known in the medical arts.

Pharmaceutical compositions of the present invention may be administeredby any route. In some embodiments, pharmaceutical compositions of thepresent invention are administered by a variety of routes, includingoral (PO), intravenous (IV), intramuscular OM), inter-arterial,intramedullary, intrathecal, subcutaneous (SQ), intraventricular,transdermal, interdermal, intradermal, rectal (PR), vaginal,intraperitoneal (IP), intragastric (IG), topical (e.g., by powders,ointments, creams, gels, lotions, and/or drops), mucosal, intranasal,buccal, enteral, vitreal, sublingual; by intratracheal instillation,bronchial instillation, and/or inhalation; as an oral spray, nasalspray, and/or aerosol; and/or through a portal vein catheter. Ingeneral, the most appropriate route of administration will depend upon avariety of factors including the nature of the agent being administered(e.g., its stability in the environment of the gastrointestinal tract),the condition of the subject (e.g., whether the subject is able totolerate a particular mode of administration), etc.

In some embodiments, vaccines in accordance with the invention aredelivered by multiple routes of administration (e.g., by subcutaneousinjection and by intranasal inhalation). For vaccines involving two ormore doses, different doses may be administered via different routes.

In some embodiments, vaccines in accordance with the invention aredelivered by subcutaneous injection. In some embodiments, vaccines inaccordance with the invention are delivered by intranasal inhalation.

In some embodiments, vaccines in accordance with the invention aredelivered by oral and/or mucosal routes. Oral and/or mucosal deliverycan prime systemic immune response. There has been considerable progressin the development of heterologous expression systems for oraladministration of antigens that stimulate the mucosal-immune system andcan prime systemic immunity. Previous efforts at delivery of oralvaccine however, have demonstrated a requirement for considerablequantities of antigen in achieving efficacy. Thus, economical productionof large quantities of target antigens is a prerequisite for creation ofeffective oral vaccines. Development of plants expressing antigens,including thermostable antigens, represents a more realistic approach tosuch difficulties.

In certain embodiments, a Y. pestis antigen expressed in a plant orportion thereof is administered to a subject orally by directadministration of a plant to a subject. In some aspects a vaccineprotein expressed in a plant or portion thereof is extracted and/orpurified, and used for preparation of a pharmaceutical composition. Itmay be desirable to formulate such isolated products for their intendeduse (e.g., as a pharmaceutical agent, vaccine composition, etc.). Insome embodiments, it will be desirable to formulate products togetherwith some or all of plant tissues that express them.

Where it is desirable to formulate product together with plant material,it will often be desirable to have utilized a plant that is not toxic tothe relevant recipient (e.g., a human or other animal). Relevant planttissue (e.g., cells, roots, leaves) may simply be harvested andprocessed according to techniques known in the art, with dueconsideration to maintaining activity of the expressed product. Incertain embodiments, it is desirable to have expressed Y. pestis antigenin an edible plant (and, specifically in edible portions of the plant)so that the material can subsequently be eaten. For instance, wherevaccine antigen is active after oral delivery (when properlyformulated), it may be desirable to produce antigen protein in an edibleplant portion, and to formulate expressed Y. pestis antigen for oraldelivery together with some or all of the plant material with which aprotein was expressed.

In some embodiments, vaccines in accordance with the invention areadministered by subcutaneous, intramuscular, and/or intravenousinjection.

In certain embodiments, Y. pestis antigens in accordance with thepresent invention and/or pharmaceutical compositions thereof (e.g.,vaccines) in accordance with the invention may be administered at dosagelevels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg,from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kgto about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about1 mg/kg to about 25 mg/kg of subject body weight per day to obtain thedesired therapeutic effect. The desired dosage may be delivered morethan three times per day, three times per day, two times per day, onceper day, every other day, every third day, every week, every two weeks,every three weeks, every four weeks, every two months, every six months,or every twelve months. In certain embodiments, the desired dosage maybe delivered using multiple administrations (e.g., two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,or more administrations).

Compositions are administered in such amounts and for such time as isnecessary to achieve the desired result. In certain embodiments, a“therapeutically effective amount” of a pharmaceutical composition isthat amount effective for treating, attenuating, or preventing a diseasein a subject. Thus, the “amount effective to treat, attenuate, orprevent disease,” as used herein, refers to a nontoxic but sufficientamount of the pharmaceutical composition to treat, attenuate, or preventdisease in any subject. For example, the “therapeutically effectiveamount” can be an amount to treat, attenuate, or prevent infection(e.g., viral infection, Y. pestis infection), etc.

It will be appreciated that Y. pestis antigens in accordance with thepresent invention and/or pharmaceutical compositions thereof can beemployed in combination therapies. The particular combination oftherapies (e.g., therapeutics or procedures) to employ in a combinationregimen will take into account compatibility of the desired therapeuticsand/or procedures and the desired therapeutic effect to be achieved. Itwill be appreciated that the therapies employed may achieve a desiredeffect for the same purpose (for example, Y. pestis antigens useful fortreating, preventing, and/or delaying the onset of Y. pestis infectionmay be administered concurrently with another agent useful for treating,preventing, and/or delaying the onset of Y. pestis infection), or theymay achieve different effects (e.g., control of any adverse effects).The invention encompasses the delivery of pharmaceutical compositions incombination with agents that may improve their bioavailability, reduceand/or modify their metabolism, inhibit their excretion, and/or modifytheir distribution within the body.

Pharmaceutical compositions in accordance with the present invention maybe administered either alone or in combination with one or more othertherapeutic agents. By “in combination with,” it is not intended toimply that the agents must be administered at the same time and/orformulated for delivery together, although these methods of delivery arewithin the scope of the invention. Compositions can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. In will be appreciated thattherapeutically active agents utilized in combination may beadministered together in a single composition or administered separatelyin different compositions. In general, each agent will be administeredat a dose and/or on a time schedule determined for that agent.

In general, it is expected that agents utilized in combination with beutilized at levels that do not exceed the levels at which they areutilized individually. In some embodiments, the levels utilized incombination will be lower than those utilized individually.

In certain embodiments, vaccine compositions comprise at least two Y.pestis antigens. For example, certain vaccine compositions can compriseat least two Y. pestis antigens in accordance with the invention (e.g.,F1 protein and/or LcrV protein). In some aspects such combinationvaccines may include one thermostable fusion protein comprising Y.pestis antigen; in some aspects, two or more thermostable fusionproteins comprising Y. pestis antigen are provided.

Where combination vaccines are utilized, it will be understood that anycombination of Y. pestis antigens may be used for such combinations.Compositions may include multiple Y. pestis antigens, including multipleantigens provided herein. Furthermore, compositions may include one ormore antigens provided herein with one or more additional antigens.Combinations of Y. pestis antigens include Y. pestis antigens derivedfrom one or more various subtypes or strains such that immunizationconfers immune response against more than one infection type.Combinations of Y. pestis antigen may include at least one, at leasttwo, at least three, at least four or more antigens derived fromdifferent subtypes or strains. In some combinations, at least two or atleast three antigens from different subtypes are combined in one vaccinecomposition. Furthermore, combination vaccines may utilize Y. pestisantigen and antigen from one or more unique infectious agents.

Pharmaceutical Compositions and/or Formulations

The present invention provides Yersinia pestis antigens andpharmaceutical compositions comprising at least one Y. pestis antigenand at least one pharmaceutically acceptable excipient (e.g., vaccinecompositions). Such pharmaceutical compositions may optionally compriseone or more additional therapeutically active substances. In accordancewith some embodiments, methods of administering a pharmaceuticalcomposition comprising administering Y. pestis antigens to a subject inneed thereof are provided. In some embodiments, pharmaceuticalcompositions are administered to humans. For the purposes of the presentdisclosure, the phrase “active ingredient” generally refers to a Y.pestis antigen in accordance with the invention. In certain embodiments,a Y. pestis antigen is or comprises F1 protein. In certain embodiments,a Y. pestis antigen is or comprises LcrV protein.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, shaping and/or packaging the product into a desired single-or multi-dose unit.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in bulk, as a single unit dose, and/oras a plurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100% (w/w) active ingredient.

Vaccines may include additionally any suitable adjuvant to enhance theimmunogenicity of the vaccine when administered to a subject. Forexample, such adjuvant(s) may include, without limitation, extracts ofQuillaja saponaria (QS), including purified subfractions of food gradeQS such as Quil A and QS-21, alum, aluminum hydroxide, aluminumphosphate, MF59, Malp2, incomplete Freund's adjuvant; complete Freund'sadjuvant, ALHYDROGEL®, 3 De-O-acylated monophosphoryl lipid A (3D-MPL).Further adjuvants include immunomodulatory oligonucleotides, for exampleunmethylated CpG sequences as disclosed in WO 96/02555. Combinations ofdifferent adjuvants, such as those mentioned hereinabove, arecontemplated as providing an adjuvant which is a preferential stimulatorof TH1 cell response. For example, QS21 can be formulated together with3 D-MPL. The ratio of QS21:3 D-MPL will typically be in the order of1:10 to 10:1; 1:5 to 5:1; and often substantially 1:1. The desired rangefor optimal synergy may be 2.5:1 to 1:1 3D-MPL: QS21. Doses of purifiedQS extracts suitable for use in a human vaccine formulation are from0.01 mg to 10 mg per kilogram of bodyweight.

It should be noted that certain thermostable proteins (e.g., lichenase)may themselves demonstrate immunoresponse potentiating activity, suchthat use of such protein whether in a fusion with a Y. pestis antigen orseparately may be considered use of an adjuvant. Thus, vaccinecompositions may further comprise one or more adjuvants. Certain vaccinecompositions may comprise two or more adjuvants. Furthermore, dependingon formulation and routes of administration, certain adjuvants may bedesired in particular formulations and/or combinations.

Pharmaceutical formulations of the present invention may additionallycomprise a pharmaceutically acceptable excipient, which, as used herein,includes any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Md.,2006) discloses various excipients used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Exceptinsofar as any conventional excipient medium is incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this invention.

In some embodiments, the pharmaceutically acceptable excipient is atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% pure: In some embodiments, the excipient is approved for use inhumans and for veterinary use. In some embodiments, the excipient isapproved by United States Food and Drug Administration. In someembodiments, the excipient is pharmaceutical grade. In some embodiments,the excipient meets the standards of the United States Pharmacopoeia(USP), the European Pharmacopoeia (EP), the British Pharmacopoeia,and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in the formulations. Excipientssuch as cocoa butter and suppository waxes, coloring agents, coatingagents, sweetening, flavoring, and/or perfuming agents can be present inthe composition, according to the judgment of the formulator.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (VEEGUM®), sodium lauryl sulfate, quaternary ammoniumcompounds, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g., acacia, agar, alginic acid,sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin,gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin),colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM®[magnesium aluminum silicate]), long chain amino acid derivatives, highmolecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleylalcohol, triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylenesorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN®60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate[SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g., CREMOPHOR®),polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [BRIJ° 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER®188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.,cornstarch, starch paste, etc.); gelatin; sugars (e.g., sucrose,glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol,etc.); natural and synthetic gums (e.g., acacia, sodium alginate,extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinylpyrrolidone), magnesium aluminum silicate [VEEGUM®], larcharabogalactan, etc.); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetlimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary anfifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONE™,KATHON™, and/or EUXYL®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., and/orcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and/or perfuming agents. In certain embodimentsfor parenteral administration, compositions are mixed with solubilizingagents such a CREMOPHOR®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or fillersor extenders (e.g., starches, lactose, sucrose, glucose, mannitol, andsilicic acid), binders (e.g., carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.,glycerol), disintegrating agents (e.g., agar, calcium carbonate, potatostarch, tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g., paraffin), absorptionaccelerators (e.g., quaternary ammonium compounds), wetting agents(e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g.,kaolin and bentonite clay), and lubricants (e.g., talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate), and mixtures thereof. In the case of capsules, tablets andpills, the dosage form may comprise buffering agents.

Solid compositions of a similar type may be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

Vaccine products, optionally together with plant tissue, areparticularly well suited for oral administration as pharmaceuticalcompositions. Oral liquid formulations can be used and may be ofparticular utility for pediatric populations. Harvested plant materialmay be processed in any of a variety of ways (e.g., air drying, freezedrying, extraction etc.), depending on the properties of the desiredtherapeutic product and its desired form. Such compositions as describedabove may be ingested orally alone or ingested together with food orfeed or a beverage. Compositions for oral administration include plants;extractions of plants, and proteins purified from infected plantsprovided as dry powders, foodstuffs, aqueous or non-aqueous solvents,suspensions, or emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oil, fish oil, andinjectable organic esters. Aqueous carriers include water, water-alcoholsolutions, emulsions or suspensions, including saline and bufferedmedial parenteral vehicles including sodium chloride solution, Ringer'sdextrose solution, dextrose plus sodium chloride solution, Ringer'ssolution containing lactose or fixed oils. Examples of dry powdersinclude any plant biomass that has been dried, for example, freezedried, air dried, or spray dried. For example, plants may be air driedby placing them in a commercial air dryer at about 120° F. until biomasscontains less than 5% moisture by weight. Dried plants may be stored forfurther processing as bulk solids or further processed by grinding to adesired mesh sized powder. Alternatively or additionally, freeze-dryingmay be used for products that are sensitive to air-drying. Products maybe freeze dried by placing them into a vacuum drier and dried frozenunder a vacuum until the biomass contains less than about 5% moisture byweight. Dried material can be further processed as described herein.

Plant-derived material may be administered as or together with one ormore herbal preparations. Useful herbal preparations include liquid andsolid herbal preparations. Some examples of herbal preparations includetinctures, extracts (e.g., aqueous extracts, alcohol extracts),decoctions, dried preparations (e.g., air-dried, spray dried, frozen, orfreeze-dried), powders (e.g., lyophilized powder), and liquid. Herbalpreparations can be provided in any standard delivery vehicle, such as acapsule, tablet, suppository, liquid dosage, etc. Those skilled in theart will appreciate the various formulations and modalities of deliveryof herbal preparations that may be applied to the present invention.

In some methods, a plant or portion thereof expressing a Y. pestisantigen according to the present invention, or biomass thereof, isadministered orally as medicinal food. Such edible compositions aretypically consumed by eating raw, if in a solid form, or by drinking, ifin liquid form. The plant material can be directly ingested without aprior processing step or after minimal culinary preparation. Forexample, a vaccine antigen may be expressed in a sprout which can beeaten directly. For instance, vaccine antigens expressed in an alfalfasprout, mung bean sprout, or spinach or lettuce leaf sprout, etc. Insome embodiments, plant biomass may be processed and the materialrecovered after the processing step is ingested.

Processing methods useful in accordance with the present invention aremethods commonly used in the food or feed industry. Final products ofsuch methods typically include a substantial amount of an expressedantigen and can be conveniently eaten or drunk. The final product may bemixed with other food or feed forms, such as salts, carriers, flavorenhancers, antibiotics, and the like, and consumed in solid, semi-solid,suspension, emulsion, or liquid form. Such methods can include aconservation step, such as, e.g., pasteurization, cooking, or additionof conservation and preservation agents. Any plant may be used andprocessed in the present invention to produce edible or drinkable plantmatter. The amount of Y. pestis antigen in a plant-derived preparationmay be tested by methods standard in the art, e.g., gel electrophoresis,ELISA, or western blot analysis, using a probe or antibody specific forproduct. This determination may be used to standardize the amount ofvaccine antigen protein ingested. For example, the amount of vaccineantigen may be determined and regulated, for example, by mixing batchesof product having different levels of product so that the quantity ofmaterial to be drank or eaten to ingest a single dose can bestandardized. A contained, regulatable environment in accordance withthe invention, however, should minimize the need to carry out suchstandardization procedures.

A vaccine protein produced in a plant cell or tissue and eaten by asubject may be preferably absorbed by the digestive system. Oneadvantage of the ingestion of plant tissue that has been only minimallyprocessed is to provide encapsulation or sequestration of the protein incells of the plant. Thus, product may receive at least some protectionfrom digestion in the upper digestive tract before reaching the gut orintestine and a higher proportion of active product would be availablefor uptake.

Dosage forms for topical and/or transdermal administration of a compoundin accordance with this invention may include ointments, pastes, creams,lotions, gels, powders, solutions, sprays, inhalants and/or patches.Generally, the active ingredient is admixed under sterile conditionswith a pharmaceutically acceptable excipient and/or any neededpreservatives and/or buffers as may be required. Additionally, thepresent invention contemplates the use of transdermal patches, whichoften have the added advantage of providing controlled delivery of acompound to the body. Such dosage forms may be prepared, for example, bydissolving and/or dispensing the compound in the proper medium.Alternatively or additionally, the rate may be controlled by eitherproviding a rate controlling membrane and/or by dispersing the compoundin a polymer matrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionsmay be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid vaccines to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes may be used in the classical mantoux method of intradermaladministration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions. Topicallyadministrable formulations may, for example, comprise from about 1% toabout 10% (w/w) active ingredient, although the concentration of theactive ingredient may be as high as the solubility limit of the activeingredient in the solvent. Formulations for topical administration mayfurther comprise one or more of the additional ingredients describedherein.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in a formulation suitable for pulmonaryadministration via the buccal cavity. Such a formulation may comprisedry particles which comprise the active ingredient and which have adiameter in the range from about 0.5 nm to about 7 nm or from about 1 nmto about 6 nm. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder and/or using a self-propelling solvent/powder dispensingcontainer such as a device comprising the active ingredient dissolvedand/or suspended in a low-boiling propellant in a sealed container. Suchpowders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nm mad at least 95% of theparticles by number have a diameter less than 7 nm. Alternatively, atleast 95% of the particles by weight have a diameter greater than 1 nmand at least 90% of the particles by number have a diameter less than 6nm. Dry powder compositions may include a solid fine powder diluent suchas sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1% to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions in accordance with the invention formulatedfor pulmonary delivery may provide the active ingredient in the form ofdroplets of a solution and/or suspension. Such formulations may beprepared, packaged, and/or sold as aqueous and/or dilute alcoholicsolutions and/or suspensions, optionally sterile, comprising the activeingredient, and may conveniently be administered using any nebulizationand/or atomization device. Such formulations may further comprise one ormore additional ingredients including, but not limited to, a flavoringagent such as saccharin sodium, a volatile oil, a buffering agent, asurface-active agent, and/or a preservative such asmethylhydroxybenzoate. The droplets provided by this route ofadministration may have an average diameter in the range from about 0.1nm to about 200 nm.

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 μm to 500 μm. Such a formulation is administered in the mannerin which snuff is taken, i.e., by rapid inhalation through the nasalpassage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition in accordancewith the invention may be prepared, packaged, and/or sold in aformulation suitable for buccal administration. Such formulations may,for example, be in the form of tablets and/or lozenges made usingconventional methods, and may, for example, 0.1% to 20% (w/w) activeingredient, the balance comprising an orally dissolvable and/ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder and/or an aerosolized and/oratomized solution and/or suspension comprising the active ingredient.Such powdered, aerosolized, and/or aerosolized formulations, whendispersed, may have an average particle and/or droplet size in the rangefrom about 0.1 nm to about 200 nm, and may further comprise one or moreof the additional ingredients described herein.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in a formulation suitable for ophthalmicadministration. Such formulations may, for example, be in the form ofeye drops including, for example, a 0.1/1.0% (w/w) solution and/orsuspension of the active ingredient in an aqueous or oily liquidexcipient. Such drops may further comprise buffering agents, salts,and/or one or more other of the additional ingredients described herein.Other opthalmically-administrable formulations which are useful includethose which comprise the active ingredient in microcrystalline formand/or in a liposomal preparation. Ear drops and/or eye drops arecontemplated as being within the scope of this invention.

In certain situations, it may be desirable to prolong the effect of avaccine by slowing the absorption of one or more components of thevaccine product (e.g., protein) that is subcutaneously orintramuscularly injected. This may be accomplished by use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The late of absorption of product then depends upon its rateof dissolution, which in turn, may depend upon size and form.Alternatively or additionally, delayed absorption of a parenterallyadministered product is accomplished by dissolving or suspending theproduct in an oil vehicle. Injectable depot forms are made by formingmicrocapsule matrices of protein in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of product topolymer and the nature of the particular polymer employed, rate ofrelease can be controlled. Examples of biodegradable polymers includepoly(orthoesters) and poly(anhydrides). Depot injectable formulationsmay be prepared by entrapping product in liposomes or microemulsions,which are compatible with body tissues. Alternative polymeric deliveryvehicles can be used for oral formulations. For example, biodegradable,biocompatible polymers such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid, etc.,can be used. Antigen(s) or an immunogenic portions thereof may beformulated as microparticles, e.g., in combination with a polymericdelivery vehicle.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005.

Kits

In some embodiments, the present invention provides pharmaceutical packsor kits including Yersinia pestis antigens according to the presentinvention. In certain embodiments, pharmaceutical packs or kits includelive sprouted seedlings, clonal entity or plant producing a Y. pestisantigen according to the present invention, or preparations, extracts,or pharmaceutical compositions containing vaccine in one or morecontainers filled with optionally one or more additional ingredients ofpharmaceutical compositions in accordance with the invention. In someembodiments, pharmaceutical packs or kits include pharmaceuticalcompositions comprising purified Y. pestis antigen according to thepresent invention, in one or more containers optionally filled with oneor more additional ingredients of pharmaceutical compositions inaccordance with the invention. In certain embodiments, thepharmaceutical pack or kit includes an additional approved therapeuticagent (e.g., Y. pestis antigen, Y. pestis vaccine) for use as acombination therapy. Optionally associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceutical products, which noticereflects approval by the agency of manufacture, use, or sale for humanadministration.

Kits are provided that include therapeutic reagents. As but onenon-limiting example, Y. pestis vaccine can be provided as oralformulations and administered as therapy. Alternatively or additionally,Y. pestis vaccine can be provided in an injectable formulation foradministration. In some embodiments, Y. pestis vaccine can be providedin an inhalable formulation for administration. Pharmaceutical doses orinstructions therefor may be provided in the kit for administration toan individual suffering from or at risk for Y. pestis infection.

The representative examples that follow are intended to help illustratethe invention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the exampleswhich follow and the references to the scientific and patent literaturecited herein. The following examples contain information,exemplification and guidance, which can be adapted to the practice ofthis invention in its various embodiments and the equivalents thereof.

EXEMPLIFICATION Example 1 A Plant-Produced Plague Vaccine CandidateConfers Protection to Monkeys

Y. pestis proteins F1 and LcrV were independently fused to an engineeredversion of the thermostable enzyme lichenase (LicKM) from Clostridiumthermocellum (Musiychuk et al., 2007, Influenza Other Respir. Viruses,1:19-25; incorporated herein by reference). Fusions were produced inNicotiana benthamiana and evaluated in Cynomolgus Macaques forimmunogenicity and protective efficacy. When administered to monkeys, amixture of the LicKM fusions to F1 and LcrV was highly immunogenic andprotective.

Materials and Methods

Engineering, Expression and Purification of Y. pestis Antigens

The LicKM fusion system for producing antigens in plants is described(Musiychuk et al., 2007, Influenza Other Respir. Viruses, 1:19-25;incorporated herein by reference). Briefly, sequence encodingfull-length mature Y. pestis F1 and LcrV were separately cloned intoLicKM (GenBank accession number DQ776900) as in-frame fusions to obtainLicKM-F1 and LicKM-LcrV. LicKM-F1 and LicKM-LcrV were individuallycloned in the plant expression vector pBID4 to give pBID4-LicKM-F1 andpBID4-LicKMLcrV; respectively, which were then separately introducedinto the Agrobacterium rhizogenes strain A4. To produce each targetantigen, A. rhizogenes strains carrying pBID4-LicKM-F I andpBID4-LicKM-LcrV were inoculated into N. benthamiana, and leaf tissuewas harvested 5 days later. Target antigens were purified fromhomogenized leaves by affinity chromatography followed by ion exchangechromatography. Purified antigens were characterized by SDS-PAGEfollowed by immunoblotting. To provide control material, LicKM alone wassimilarly expressed in and purified from N. benthamiana.

Cynomolgus Macaques Challenge Study Using Plant-Produced Y. pestisAntigens

The study was conducted using female Cynomolgus Macaques (CovanceResearch Products) of approximately 2 years of age and approximately 2kg weight. For test groups, LicKM-F1 and LicKM-LcrV were mixed at aweight ratio of 1:1 to give the candidate vaccine (CV). Where antigenswere to be delivered with adjuvant, they were mixed with 2% ALHYDROGEL®(Accurate Chemical & Scientific Corporation) at a ratio of 1:50 (w/w;antigen/adjuvant). The study comprised four groups: Group 1 (negativecontrol) had two animals and groups 2-4 had three animals per group.Group 1 received 125 μg/dose of LicKM plus adjuvant. Group 2 received 25μg/dose of CV plus adjuvant. Group 3 received 250 μg/dose of CV plusadjuvant, and group 4 received 250 μg/dose of CV alone. Antigens wereadministered by subcutaneous injection on study days 1, 14, and 28.Serum samples were collected on days of candidate vaccine administrationand 7 days after the final administration. Animals were challenged vianose-only inhalation with Y. pestis strain CO 92; Biovar-Orientalis at100×LD₅₀ on study day 40 and observed for a further 14 days.

Analysis of Serum Samples for Immune Responses to Administered Antigens

Sera collected from immunized monkeys were analyzed for the presence ofLcrV and F1-specific IgG and IgA by ELISA. MaxiSorp 96-well plates(Nunc) were coated with 1 μg/ml Escherichia coli-produced F1 fused todomain 1 of Bacillus anthracis lethal factor (LFD1) or E. coli-producedLcrV. Serum samples were added at an initial dilution of 1:100, titratedin five-fold dilutions, and target-specific antibodies were detectedusing goat anti-monkey IgG (KPL) or IgA (Fitzgerald IndustriesInternational Inc.) conjugated to HRP.

Analysis of Tissue Pathogen Load

Tissues from all challenged animals were evaluated for presence of Y.pestis. Tissues were placed in 1% peptone and individually homogenized.Tissue homogenates were serially diluted in 1% peptone, and 100 μlaliquots were spread plated on 90 mm tryptic soy agar (TSA) plates intriplicate. TSA plates were incubated at 28° C. for 36 h-48 h, afterwhich Y. pestis colonies were counted. Pathogen load is expressed ascolony forming units (CFU).

Results

Expression of Y. pestis FI and LcrV Antigens as Fusions to LicKM in N.benthamiana

LicKM, LicKM-F1, and LicKM-LcrV were purified from N. benthamiana leaftissue and analyzed by SDS-PAGE and immunoblot (FIG. 2). Gels werestained with Coomassie Brilliant Blue to show purified LicKM, LicKM-F1,and LicKM-LcrV (FIG. 2A). On average, 380 μg LicKM-F1 and 120 μg ofLicKM-LcrV was purified per gram of fresh leaf tissue. In immunoblotassays, antibodies specific for LicKM reacted with LicKM and both fusionproteins (FIG. 2B), whereas antibodies specific for either LcrV (FIG.2C) or F1 (FIG. 2D) reacted only with their respective LicKM fusionproteins.

Immunogenicity and Protective Efficacy of Plant-Produced F1 and LcrV

To evaluate immunogenicity and protective efficacy of plant-producedantigens, animals wore immunized with a mixture of LicKM-F1 andLicKM-LcrV or with LicKM alone. Serum samples were assessed for thepresence of IgG and IgA specific to LcrV and F1. All animals in group 3mounted a strong IgG response against both LcrV (FIG. 3A) and F1 (FIG.3B). IgG antibody titers against LcrV approached peak values followingthe priming dose mad did not substantially increase following boosterdoses. In group 4, which received the same dose of antigen but in theabsence of adjuvant, IgG responses specific to LcrV were up to two logslower than group 3 following the priming dose and remained significantlylower than group 3 even after booster doses (FIG. 3A). Also, the IgGresponse to F1 in group 4 was negligible, even after the two boosts(FIG. 3B). These results indicate that adjuvant can help stimulate hightiter antibody responses. Animals in group 2 that were immunized with10-fold less antigen in the presence of adjuvant produced anti-LcrVantibodies with titers as high as group 3 (FIG. 3A). However,F1-specific IgG titers in this group were approximately two logs lowerthan group 3 (FIG. 3B). Production of serum LcrV-specific IgA wasdetected at similar levels in animals in groups 2 and 3 and peaked afterthe prime (FIG. 3C). Group 4 animals produced detectable amounts ofserum IgA against LcrV (FIG. 3C), although at lower titers than observedin groups 2 and 3. In all test groups, F1-specific serum IgA responseswere lower than LcrV-specific IgA responses and were not measurable inall animals (FIG. 3D). No LcrV- or F1-specific antibodies were detectedin control animals.

Following immunization, vaccinated animals were challenged withaerosolized Y. pestis. All animals in group 1 developed clinical signsof disease and succumbed to death 5 days after challenge (FIG. 3E). Bycontrast, all animals in group 3 survived the challenge, indicating thatthe plant-produced LicKMF1/LicKM-LcrV antigen mixture is fullyprotective. Two of the three animals in group 2 survived the challengebut, none of the animals in group 4 survived (FIG. 3E). Post-mortemanalysis of pathogen load in different organs of animals that survivedthe challenge revealed no Y. pestis, whereas organs collected fromanimals that died of challenge had high titers of bacteria (Table 2).

TABLE 2 Tissue Pathogen Load (CFU) in Monkeys Following Y. pestisChallenge Group Spleen Liver Lymph Node Lung 1 >2 × 10⁶ >2.5 × 10⁶  >6.8× 10⁶ >7.4 × 10⁶ >2 × 10⁶  >2 × 10⁶ >1.1 × 10⁷ >2.3 × 10⁶ 2 >2 × 10⁶  >2× 10⁶  >2 × 10⁶  >2 × 10⁶ 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 43.3 × 10⁶  5.8 × 10⁶  3.6 × 10⁸  >4 × 10⁹ >2 × 10⁶ >3.1 × 10⁶  >9.9 ×10⁶ >2.9 × 10⁶  2 × 10⁹ 4.4 × 10⁸  1.3 × 10⁹ >8.3 × 10⁹

In summary, plant-produced antigens stimulated strong antibody responsesand provided full protection against challenge with aerosolized Y.pestis in primates. The present invention encompasses the recognitionthat plant-produced Y. pestis antigens may stimulate strong antibodyresponses and provide full or partial protection against Y. pestisinfection in humans, non-human primates, and other mammals (e.g., cats,dogs, mice, rats, horses, cows, etc.).

Example 2 A Plant-Produced Plague Double Fusion Vaccine CandidateStimulates High Titers of Antigen-Specific IgG and Confers Protection toMammals

Y. pestis proteins F1 and LcrV were both fused to an engineered versionof the thermostable enzyme lichenase (LicKM) from Clostridiumthermocellum (Musiychuk et al., 2007, Influenza Other Respir. Viruses,1:19-25; incorporated herein by reference). LcrV protein was fused intothe loop region of LicKM, and F1 protein was fused to the C-terminus ofLicKM. Fusions were produced in Nicotiana benthamiana, and serum LcrV-and F1-specific IgG titers were measured. Fusions were also evaluated inCynomolgus Macaques for immunogenicity and protective efficacy. When‘administered to monkeys, the double fusion generated high LcrV- andF1-specific IgG liters, and the double fusion was found to be highlyimmunogenic and protective.

Engineering, Expression, and Purification of Y. pestis Double FusionAntigen

The LicKM fusion system for producing antigens in plans is described(Musiychuk et al., 2007, Influenza Other Respir. Viruses, 1:19-25;incorporated herein by reference). Briefly, science encoding full-lengthmature Y. pestis F1 and LcrV were both cloned into LicKM (GenBankaccession number DQ77690) as in-frame fusions to obtain LcrV-F1-LicKM.LcrV was cloned into the loop region of LicKM, and F1 was fused to theC-terminus of LicKM.

The nucleotide sequence of the double fusion construct, which encodesdouble fusion protein antigen, is:

(SEQ ID NO: 11) 5′GGTACC

ATGGGTTTCGTGCTTTTCTCTCAG CTTCCTTCTTTCCTTCTTGTGTCTACCCTTCTTCTTTTCCTTGTGATTTC TCACTCTTGCAGGGCT CAGAATGGTGGTTCTTACCCTTACAAGTCTGGTGAGTACAGGACCAAGTCTTTCTTCGGTTACGGTTACTACGAAGTGAGAATGAAGGCTGCTAAGAATGTGGGTATTGTGTCATCTTTCTTCACCTACACCGGTCCTTCAGATAATAACCCTTGGGATGAGATTGATATTGAGTTCCTTGGAAAGGATACCACCAAGGTTCAGTTCAACTGGTACAAGAACGGTGTTGGTGGAAATGAGTACCTTCACAACCTTGGTTTCGATGCTTCTCAGGATTTCCACACCTACGGTTTTGAGTGGAGGCCTGATTACATTGATTTCTACGTGGATGGAAAGAAGGTTTACAGGGGAACCAGGAACATTCCTGTTACCCCTGGAAAGATTATGATGAACCTTTGGCCTGGTATTGGTGTTGATGAGTGGCTTGGTAGATACGATGGAAGGACTCCTCTTCAGGCTGAGTACGAGTACGTTAAGTACTACC CTAACGGT

ATGATTAGGGCTTA CGAGCAGAATCCTCAGCAC TTCATTGAGGATCTTGAGAAAGTTAGGGTGGAGCAGCTTACTGGTCATGG TTCTTCAGTGCTTGAAGAGCTTGTTCAGCTTGTGAAGGATAAGAACATTG ATATTTCTATTAAGTACGATCCTAGGAAGGATTCTGAGGTGT TCGCTAACAGAGTGATTACCGATGATATTGAGCTTCTGAAGAAGATTCTTGCTT ACTT CCTTCCTGAGGATGCTATTCTTAAGGGTGGTCACTACGATAATCAGCTTCAGAACGGTATTAAGAGGGTGAAAGAGTTCCTTGAGTCATCTCCTAATACC CAGTGGGAGCTTAGGGCTTTTATGGCTGTGATGCACTTCTCTCTTACCGC TGATAGGATTGATGATGATATTCTTAAAGTGATTGTGGATTCTATGAACC ACCATGGTGATGCTAGGTCTAAGTTGAGGGAAGAGCTTGCTGAACTTACC GCTGAGTTGAAAATCTACTCTGTGATTCAGGCTGAGATTAACAAGCACCT TTCTTCATCTGGAACCATTAACATTCACGATAAGTCTATTAACCTTATGG ATAAGAACCTTTACGGTTACACCGATGAAGAGATTTTCAAGGCTTCTGCT GAGTACAAGATTCTTGAGAAGATGCCTCAGACTACCATTCAAGTGGATGG TTCTGAGAAGAAAATTGTGTCTATTAAGGATTTCCTTGGATCTGAGAACA AGAGGACTGGTGCTTTGGGTAACCTTAAGAACTCT TACTCTTACAACAAGGATAACAACGAGCTTTCTCACTTCGCTACTACCTGCTCT GATAAGTCTAGGCCTCTTAACGATCTTGTGTCTCAAAAGACCACCCAGCTTTCT GATATTACCTCTAGGTTCAACTCTGCTATTGAGGCTCTTAACAGATTCATTCAG AAATACGATTCTGTGATGCAAAGGCTTCTTGATGATACCTCTGGAAAG

GTTGTTAACACCCCTTTCGTGGCTGTTTTCTCTAACTTCGATTCTTCTCAGTGGGAAAAGGCTGATTGGGCTAACGGTTCTGTGTTCAACTGTGTGTGGAAGCCTTCTCAGGTGACCTTCTCTAACGGAAAGATGATTCTTACCCTTGATAGGGAATACGTCG

GCTGATT TGACTGCTTCTACTACT GCTACTGCTACTCTTGTTGAGCCTGCTAGGATTACCCTTACCTACAAAGA GGGTGCTCCTATTACTATTATGGATAACGGTAACATTGATA CCGAGTTGCTTGTGGGTACTCTTACACTTGGTGGTTACAAGACCGGTACTACCT CTACTTCTGTGAACTTCACCGATGCTGCTGGTGATCCTATGTACCTTACCTTCAC CTCTCAGGATGGAAATAACCACCAGTTCACCACCAAAGTGATTGGAAAGG ATTCTAGGGATTTCGATATTTCTCCTAAGGTGAACGGTGAAAATCTTGTG GGTGACGATG TTGTTCTTGCTACCGGTTCACAGGATTTCTTTGTGAGATC AATTGGTTCTAAGGGTGGAAAGTTGGCTGCTGGAAAGTACACTGATGCTG TGACTGTGAC TGT GTCTAATCAG

CATCATCATCATCACCAC AAGGATGAGCTTTGATGACTCGAGCTC 3′.The plain text sequences correspond to LicKM sequences (e.g., CAG . . .GGT, SEQ ID NO: 12; and GGT . . . TAC, SEQ ID NO: 13), a 6×-His tag(i.e., 5′ CATCATCATCATCACCAC 3′ SEQ ID NO: 14), and an ER retentionsignal (i.e., 5′ AAGGATGAGCTT 3′ SEQ ID NO: 15). The bold, underlinedsequence ATG . . . GCT (SEQ ID NO: 16) corresponds to the PR1a signalpeptide. The bold, underlined sequence ATG . . . AAG (SEQ ID NO: 17)corresponds to LcrV protein coding sequence. The bold, underlinedsequence GCT . . . CAG (SEQ ID NO: 18) corresponds to FI protein codingsequence. The bold, italicized sequence GGATCCTTAATTAA (SEQ ID NO: 19)corresponds to a BamHI site (i.e., GGATCC, SEQ ID NO: 20) and a Pad site(i.e., TTAATTAA, SEQ ID NO: 18). The bold, italicized sequence AGACTC(SEQ ID NO: 21) corresponds to a BglII site. The bold, italicizedsequence AAGCTT (SEQ ID NO: 22) corresponds to a HindIII site. The bold,italicized sequences GTCGAC (SEQ ID NO: 23) correspond to two SalIsites. The bold, italicized sequence CTCGAGCTC (SEQ ID NO: 24)corresponds to an XhoI site (i.e., CTCGAG, SEQ ID NO: 25) and a Sad site(i.e., GAGCTC, SEQ ID NO: 26).

The amino acid sequence of the double fusion protein antigen is:

(SEQ ID NO: 27) MGFVLFSQLPSFLLVSTLLLFLVISHSCRA QNGGSYPYKSGEYRTKSFFGYGYYEVRMKAAKNVGIVSSFFTYTGPSDNNPWDEIDIEFLGKDTTKVQFNWYKNGVGGNEYLHNLGFDASQDFHTYGFEWRPDYIDFYVDGKKVYRGTRNIPVTPGKIMMNLWPGIGVDEWLGRYDGRTPLQAEYEYVKYYPNG

MI RAYEQNPQIIFIEDLEKVRVEQLTGHGSSVLEEL VQLVKDKNIDISIKY DPRKDSEVFANRVITDDIELLKKILAYFLPEDAILKGGHYDNQ LQNGIK RVKEFLESSPNTQWELRAFMAVMITFSLTADRIDDDILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKS1NLMDKNLYG YTDEEIFKASAEYKILEKMPQTTIQVDGSEKKWSIKDFLGSENKRTGALG NLKNSYSYNKDNNELSIIFATTCSDKSRPLNDLVSQKTTQLSDITSRFNS AIEALNRFIQKYDSVMQRLL DDTSGK

VVNTPFVAVFSNFDSSQWEK ADWANGSVFNCVWKPSQVTFSNGKMILTLDREY

ADLTASTTATATL VEPARITLTYKEGAPITIMDNGNIDTELLVGTLTLGGY KTGTTSTSVNFTDAAGDPMYLTFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENL VGDDVVLATGSQDFFVRSIGSKGGKILAAGKYTDAVTVTVSNQ

HHHHHHKDE L 3′.The plain text sequences correspond to LicKM sequences (e.g., QNG . . .PNG, SEQ ID NO: 28; and VVN . . . REY, SEQ ID NO: 29), a 6×-His tag(i.e., HHHHHH, SEQ ID NO: 30), and an ER retention signal (i.e., KDEL;SEQ ID NO: 31). The bold, underlined sequence MGF . . . CRA (SEQ ID NO:32) corresponds to the PR1a signal peptide. The bold, underlinedsequence MIR . . . SGK (SEQ ID NO: 33) corresponds to LcrV proteincoding sequence. The bold, underlined sequence ADL . . . SNQ (SEQ ID NO:34) corresponds to F1 protein coding sequence. The bold, italicizedsequence RS (SEQ ID NO: 35) corresponds to a BglII site. The bold,italicized sequence KL (SEQ ID NO: 36) corresponds to a HindIII site.The bold, italicized sequences VD (SEQ ID NO: 37) correspond to two SalIsites.

To provide control material, LicKM alone was similarly expressed in andpurified from N. benthamiana.

LicKM-F1 and LicKM-LcrV were individually cloned in the plant expressionvector pGREENII to give pGREEN-LcrV-F1-LicKM, which was introduced intoAgrobacterium rhizogenes. A. rhizogenes were inoculated into N.benthamiana, and leaf tissue was harvested (e.g., about 5 days later).Target antigens were purified from homogenized leaves by chromatographysteps (e.g., affinity chromatography followed by ion exchangechromatography). Purified antigens were characterized by Coomassiebrilliant blue staining and by SDS-PAGE followed by immunoblotting. Toprovide control material, LicKM alone was similarly expressed in andpurified from N. benthamiana.

Cynomolgus Macaques Challenge Study Using Plant-Produced Y. pestisAntigens

Five groups of female monkeys (five in Groups 1 and 3, eight in Group 2and 4, and four in Group 5) each received either a vaccination asoutlined in Table 3. Monkeys received each dose on Study Days 1, 14, and28 using modes of administration specified in Table 3.

TABLE 3 Double Fusion Vaccine Administration Vaccine Route andComposition Dose Frequency Group 1 LicKM alone 125 μg + subcutaneousALHYDROGEL ® + injection, thrice QuilA Group 2 LicKM-F1 + 250 μg +subcutaneous LicKM-LcrV ALHYDROGEL ® + injection, thrice QuilA Group 3Lic KM alone (a) 125 μg + (a) subcutaneous, ALHYDROGEL ® + once QuilA(b) intranasal, (b) 125 μg second and third (no adjuvant) Group 4LicKM-F1 + (a) 125 μg + (a) subcutaneous, LicKM-LcrV ALHYDROGEL ® + onceQuilA (b) intranasal, (b) 125 μg second and third (no adjuvant) Group 5LicKM-F1 + 250 μg + subcutaneous LicKM ALHYDROGEL ® + injection, thricedouble fusion QuilAAll monkeys were challenged via inhalation with a multiple LDs0inhalation dose of Y. pestis on Study Day 40. Monkeys were evaluated for14 days post pathogen challenge for disease development and mortality.Evaluations during the study included twice daily clinical observationsand qualitative assessment of food consumption. Body weights wereobtained at predetermined times during the experiment as were bloodpressure measurements and radiographs. A physical examination of eachmonkey was conducted by a licensed veterinary technician under thesupervision of a veterinarian prior to initiation of vaccination, beforepathogen challenge, and prior to euthanasia. Subcutaneous bodytemperatures were obtained twice daily during the study beginning onStudy Day 7. Serum samples were obtained periodically to assess antibodytiters. Vaginal wash specimens were obtained periodically during thestudy to assess mucosal antibody titers. Clinical pathology (e.g.,hematology and serum chemistry) was assessed periodically pre- andpost-challenge. Y. pestis load was determined in whole blood at definedintervals. Selected tissues from dead or euthanized animals were alsoevaluated for Y. pestis load. Tissue specimens were obtained from allanimals and preserved in 10% buffered formalin. Selected tissues wereevaluated for histopathology.

Results and Discussion

Expression of Y. pestis F1 and LcrV Antigens as a Double Fusion to LicKM

LcrV-F1-LicKM was purified from N. benthamiana leaf tissue and analyzedby SDS-PAGE and immunoblot (FIG. 4, lanes 5-8). Gels were stained withCoomassie Brilliant Blue to show purified LcrV-F1-LicKM (FIG. 4, lanes1-4). In immunoblot assays, antibodies specific for LicKM reacted withLicKM, with the double fusion protein, and with a fusion of LicKM to anunrelated protein (i.e., anthrax lethal factor (LF) protein) (FIG. 4).

Immunogenicity and Protective Efficacy of Plant-Produced F1-LcrV-LicKM

To evaluate immunogenicity and protective efficacy of plant-producedF1-LcrV-LicKM double fusion antigens, animals were immunized with thedouble fusion, with a mixture of LicKM-F1 and LicKM-LcrV, or with LicKMalone (see Table 3). Serum samples were assessed at Study Days −9 (i.e.,9 days prior to the first immunization dose), 14, 28, and 35 for thepresence of IgG specific to LcrV and F1. All animals in Group 2 (i.e.,mixture of LicKM-F1 and LicKM-LcrV plus two adjuvants administeredthrice by subcutaneous injection) and Group 5 (i.e., F1-LcrV-LicKMdouble fusion plus two adjuvants) mounted strong IgG responses againstboth LcrV (FIGS. 5A and 5C) and F1 (FIGS. 5B and 5D). Immune responsesmounted by animals in Group 1 (i.e., LicKM alone plus two adjuvants)were 1-2 logs lower than those mounted by animals in Group 2 or Group 5after the first immunization dose.

Following immunization, vaccinated animals were challenged withaerosolized Y. pestis. All five animals in Group 1 developed clinicalsigns of disease and succumbed to death or were considered moribund (andwere, therefore, euthanized) within 9 days after challenge (i.e., about0% survival). By contrast, seven of eight animals in Group 2 survivedthe challenge (i.e., about 88% survival). Three of four monkeys in Group3 died (i.e., about 25% survival). Five of eight monkeys in Group 4 werefound dead or were considered moribund (and were, therefore, euthanized)within 6 days after challenge (i.e., about 38% survival). All fivemonkeys in Group 5 survived to the end of the study (i.e., about 100%survival). Survival data are summarized in FIG. 6.

At study initiation, all monkeys were below the level of detection forY. pestis. Post-pathogen exposure, all monkeys that did not survive tostudy termination were bacteremic. Group 1: By post-immunization day 2bacteria was detected in one of five monkeys and by post-immunizationday 3 two of five were bacteremic. Bacteria were not detected in theblood of the remaining three monkeys although as discussed below,pathogen was cultured from their tissues. Group 2: Only one monkey inthis group did not to survive to study termination. Bacteria were notfound in its blood, but there was substantial tissue tropism. Theremaining monkeys in this group had pathogen levels below the detectablelevel (except one monkey displayed small but transient presence onpost-immunization day 4). Group 3: Three of the four monkeys exposed tothe pathogen had detectable blood levels of Y. pestis. One monkey had nodetectable levels of Y. pestis. Pathogen was not detected in any of thetissues taken at study termination. Group 4: The five monkeys that didnot survive to the end & the study had bacteria in their blood, and thethree monkeys that survived to study termination were below the level ofpathogen detection. Group 5: All four monkeys in this group survived tostudy termination with no detectable blood levels of pathogen.

All monkeys that did not survive to the end of the study (i.e., monkeysthat did not survive to post-immunization day 14) exhibited tissuepathogen loads in all of the tissues evaluated. Pathogen tropism wasmost evident for lymph nodes and lung. For monkeys that survived topost-immunization day 14, there was no detectable pathogen in theevaluated tissues. In summary, plant-produced F1-LcrV-LicKM doublefusion protein antigens stimulated strong antibody responses andprovided 100% protection against challenge with aerosolized Y. pestis inprimates. The present invention encompasses the recognition that resultsobtained in mammals (e.g., primates) can be predictive of therapeuticand/or prophylactic efficacy in humans. The present inventionencompasses the recognition that plant-produced Y. pestis antigens maystimulate strong antibody responses and provide full or partialprotection against Y. pestis infection in humans, non-human primates,and other mammals (e.g., cats, dogs, mice, rats, horses, cows, etc.).

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention, described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process. Furthermore, it is to be understood that theinvention encompasses all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim. For example, any claim that is dependent on another claim can bemodified to include one or more limitations found in any other claimthat is dependent on the same base claim. Furthermore, where the claimsrecite a composition, it is to be understood that methods of using thecomposition for any of the purposes disclosed herein are included, andmethods of making the composition according to any of the methods ofmaking disclosed herein or other methods known in the art are included,unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldit be understood that, in general, where the invention, or aspects ofthe invention, is/are referred to as comprising particular elements,features, etc., certain embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements, features,etc. For purposes of simplicity those embodiments have not beenspecifically set forth in haec verba herein. It is noted that the term“comprising” is intended to be open and permits the inclusion ofadditional elements or steps.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., any Y.pestis strain; any Y. pestis protein; any fusion protein; any expressionsystem; any plant production system; any method of administration; etc.)can be excluded from any one or more claims, for any reason, whether ornot related to the existence of prior art.

What is claimed is:
 1. An isolated antigen comprising two or moreYersinia proteins fused to a LicKM protein, wherein a first of the twoor more Yersinia proteins comprises a full-length F1 protein that is atleast 95% identical to the sequence set forth as SEQ ID NO:1, 3, 5, or7, and wherein a second of the two or more Yersinia proteins comprises afull-length LcrV protein that is at least 95% identical to the sequenceset forth as SEQ ID NO:9.
 2. The isolated antigen of claim 1, wherein afirst of the two or more Yersinia proteins comprises a full-length F1protein that is at least 98% identical to the sequence set forth as SEQID NO:1, 3, 5, or 7, and wherein a second of the two or more Yersiniaproteins comprises a full-length LcrV protein that is at least 98%identical to the sequence set forth as SEQ ID NO:9.
 3. The isolatedantigen of claim 1, wherein a first of the two or more Yersinia proteinscomprise the amino acid sequence set forth as SEQ ID NO:1, 3, 5, or 7,and wherein a second of the two or more Yersinia proteins comprises theamino acid sequence set forth as SEQ ID NO:9.
 4. The isolated antigen ofclaim 1, wherein the two or more Yersinia proteins are fused to theLicKM protein as an N-terminal fusion, a C-terminal fusion, a surfaceloop insertion fusion, or a combination thereof.
 5. The isolated antigenof claim 1, wherein the antigen comprises an amino acid sequence that isat least 95% identical to the sequence set forth as SEQ ID NO:27.
 6. Theisolated antigen of claim 1, wherein the antigen comprises an amino acidsequence that is at least 98% identical to the sequence set forth as SEQID NO:27.
 7. The isolated antigen of claim 1, wherein the antigencomprises the amino acid sequence set forth as SEQ ID NO:27.
 8. Avaccine composition comprising a pharmaceutically acceptable carrier andan antigen comprising two or more Yersinia proteins fused to a LicKMprotein, wherein a first of the two or more Yersinia proteins comprisesa full-length F1 protein that is at least 95% identical to the sequenceset forth as SEQ ID NO:1, 3, 5, or 7, and wherein a second of the two ormore Yersinia proteins comprises a full-length LcrV protein that is atleast 95% identical to the sequence set forth as SEQ ID NO:9, andwherein the composition is capable of eliciting an immune response uponadministration to a subject.
 9. The vaccine composition of claim 8,wherein a first of the two or more Yersinia proteins comprises afull-length F1 protein that is at least 98% identical to the sequenceset forth as SEQ ID NO:1, 3, 5, or 7, and wherein a second of the two ormore Yersinia proteins comprises a full-length LcrV protein that is atleast 98% identical to the sequence set forth as SEQ ID NO:9.
 10. Thevaccine composition of claim 8, wherein a first of the two or moreYersinia proteins comprises the amino acid sequence set forth as SEQ IDNO:1, 3, 5, or 7, and wherein a second of the two or more Yersiniaproteins comprises the amino acid sequence set forth as SEQ ID NO:9. 11.The vaccine composition of claim 8, wherein the two or more Yersiniaproteins are fused to the LicKM protein as an N-terminal fusion, aC-terminal fusion, a surface loop insertion fusion, or a combinationthereof.
 12. The vaccine composition of claim 8, wherein the antigencomprises an amino acid sequence that is at least 95% identical to thesequence set forth as SEQ ID NO:27.
 13. The vaccine composition of claim8, wherein the antigen comprises an amino acid sequence that is at least98% identical to the sequence set forth as SEQ ID NO:27.
 14. The vaccinecomposition of claim 8, wherein the antigen comprises the amino acidsequence set forth as SEQ ID NO:27.
 15. The vaccine composition of claim8, wherein the antigen is produced in a transgenic plant or in a planttransiently expressing the antigen.
 16. The vaccine composition of claim8, wherein the composition comprises an antigen that is purified orpartially purified from plant cells, a plant, seeds, fruit, or anextract thereof.
 17. The vaccine composition of claim 8, furthercomprising at least one vaccine adjuvant.
 18. The vaccine composition ofclaim 17, wherein the adjuvant is selected from the group consisting ofcomplete Freund's adjuvant, incomplete Freund's adjuvant, alum, MF59,saponin, aluminum hydroxide, QuilA, and macrophage-activatinglipopeptide (Malp-2).
 19. A method for inducing a protective immuneresponse against Yersinia infection in a subject, comprisingadministering to the subject an effective amount of an anti-Yersiniavaccine composition, wherein the administration is sufficient tostimulate production of antigen specific antibodies or stimulate acellular immune response by the subject, thereby inducing a protectiveimmune response, wherein the vaccine composition comprises an antigencomprising two or more Yersinia proteins fused to a LicKM protein,wherein a first of the two or more Yersinia proteins comprises afull-length F1 protein that is at least 95% identical to the sequenceset forth as SEQ ID NO:1, 3, 5, or 7, and wherein a second of the two ormore Yersinia proteins comprises a full-length LcrV protein that is atleast 95% identical to the sequence set forth as SEQ ID NO:9.
 20. Themethod of claim 19, wherein a first of the two or more Yersinia proteinscomprises a full-length F1 protein that is at least 98% identical to thesequence set forth as SEQ ID NO:1, 3, 5, or 7, and wherein a second ofthe two or more Yersinia proteins comprises a full-length LcrV proteinthat is at least 98% identical to the sequence set forth as SEQ ID NO:9.21. The method of claim 19, wherein a first of the two or more Yersiniaproteins comprises the amino acid sequence set forth as SEQ ID NO:1, 3,5, or 7, and wherein a second of the two or more Yersinia proteinscomprises the amino acid sequence set forth as SEQ ID NO:9.
 22. Themethod of claim 19, wherein the two or more Yersinia proteins are fusedto the LicKM protein as an N-terminal fusion, a C-terminal fusion, asurface loop insertion fusion, or a combination thereof.
 23. The methodof claim 19, wherein the antigen comprises an amino acid sequence thatis at least 95% identical to the sequence set forth as SEQ ID NO:27. 24.The method of claim 19, wherein the antigen comprises an amino acidsequence that is at least 98% identical to the sequence set forth as SEQID NO:27.
 25. The method of claim 19, wherein the antigen comprises theamino acid sequence set forth as SEQ ID NO:27.
 26. The method of claim19, wherein the composition is administered orally, intranasally,subcutaneously, intravenously, intraperitoneally, or intramuscularly.27. The method of claim 19, wherein the composition comprises plantcells containing the antigen, and wherein the administration comprisesorally via feeding plant cells to the subject.
 28. The method of claim19, wherein the subject is human.
 29. A composition comprising a firstisolated antigen and a second antigen, wherein the first antigencomprises a first Yersinia protein fused to a LicKM protein, wherein thefirst Yersinia protein comprises a full-length F1 protein that is atleast 95% identical to the sequence set forth as SEQ ID NO:1, 3, 5, or7, wherein the second antigen comprises a second Yersinia protein fusedto a LicKM protein, and wherein the second Yersinia protein comprises afull-length LcrV protein that is at least 95% identical to the sequenceset forth as SEQ ID NO:9.
 30. The composition of claim 29, wherein thefirst Yersinia protein comprises the amino acid sequence set forth asSEQ ID NO:1, 3, 5, or 7, and wherein the second Yersinia proteincomprises the amino acid sequence set forth as SEQ ID NO:9.